Pregnancy-associated plasma protein-A2 (PAPP-A2) polynucleotides

ABSTRACT

The present invention provides pregnancy associated plasma protein A2 (PAPP-A2), its nucleotide and amino acid sequences, antisense molecules to the nucleotide sequences which encode PAPP-A2, expression vectors for the production of purified PAPP-A2, antibodies capable of binding specifically to PAPP-A2, hybridization probes or oligonucleotides for the detection of PAPP-A2-encoding nucleotide sequences, genetically engineered host cells for the expression of PAPP-A2, and methods for screening for pathologies in pregnant and non-pregnant patients. Methods for screening for altered focal proliferation states in pregnant and/or non-pregnant patients, which include detecting levels of PAPP-A2, are also described.

FIELD OF THE INVENTION

The present invention relates to a novel polypeptide with homology topregnancy-associated plasma protein-A (PAPP-A). The novel polypeptideaccording to the invention is denoted PAPP-A2. The invention furtherrelates to novel polynucleotides comprising a nucleic acid sequenceencoding such a polypeptide, or a fragment thereof.

The invention further relates to methods for using the novelpolynucleotides, including fragments thereof as defined herein below,and methods for using the novel polypeptides capable of being producedfrom such polynucleotides.

The invention also relates to expression and purification of recombinantPAPP-A2, and to production of polyclonal and monoclonal antibodiesagainst PAPP-A2, and to the purification of native PAPP-A2 from humantissues or body fluids.

In further aspects the invention relates to uses of PAPP-A2 as a markerfor pathological states, and as a therapeutic target for drugs thatmodify the proteolytic activity of PAPP-A2 in pregnant as well asnon-pregnant individuals.

BACKGROUND OF THE INVENTION

Pregnancy-Associated Plasma Protein-A (PAPP-A)

PAPP-A was first isolated in 1974 from pregnancy serum along with otherproteins believed to be of placental origin (Lin et al., 1974, Am JObstet Gynecol 118, 223-36). The concentration in serum reaches about 50mg/liter at the end of pregnancy (Folkersen et al., 1981, Am J ObstetGynecol 139, 910-4; Oxvig et al., 1995, J Biol Chem 270, 13645-51).PAPP-A was originally characterized as a high molecular weighthomotetramer (Bischof, 1979, Arch Gynecol 227, 315-26; Lin et al., 1974,Am J Obstet Gynecol 118, 223-36; Sinosich, 1990, Electrophoresis 11,70-8), but it has now been demonstrated that PAPP-A primarily exists inpregnancy serum and plasma as a covalent, heterotetrameric 2:2 complexwith the proform of eosinophil major basic protein (proMBP),PAPP-A/proMBP (Oxvig et al., 1993, J Biol Chem 268, 12243-6). Only about1% of PAPP-A in pregnancy serum and plasma is present as a homodimer, asrecently demonstrated (Overgaard et al., 2000, J Biol Chem). Theexistence of the PAPP-A/proMBP complex was revealed, in part, by theisolation of a PAPP-A and a proMBP peptide, linked together by adisulfide bond, from a digest of purified PAPP-A/proMBP (Oxvig et al.,1993, J Biol Chem 268, 12243-6).

The subunits of the PAPP-A/proMBP complex can be irreversibly separatedby reduction of disulfide bonds and denaturation (Oxvig et al., 1993, JBiol Chem 268, 12243-6). In reducing SDS-PAGE, the PAPP-A subunit has anapparent molecular weight of 200 kDa (Oxvig et al., 1994, BiochimBiophys Acta 1201, 415-23), and its 1547-residue sequence is known fromcloned cDNA (Kristensen et al., 1994, Biochemistry 33, 1592-8). PAPP-Ais synthesized as a pre-pro-protein (preproPAPP-A), including a80-residue pre-pro-piece (Haaning et al., 1996, Eur J Biochem 237,159-63). No proteins with global homology to PAPP-A has been reported inthe literature, but PAPP-A contains sequence motifs, including anelongated zinc binding motif (HEXXHXXGXXH) (SEQ ID NO:26) at position482-492 (numbering according to Kristensen et al., 1994, Biochemistry33, 1592-8). This motif and a structurally important methionine residue,also thought to reside in PAPP-A at position 556, are strictly conservedwithin the metzincins, a superfamily of zinc peptidases: astacins,adamalysins (or reprolysins), serralysins and matrixins (matrixmetalloproteinases or MMP's) (Bode et al., 1993, FEBS Lett 331, 134-40;Stocker et al., 1995, Protein Sci 4, 823-40).

The proMBP subunit has a calculated peptide mass of 23 kDa (Barker etal., 1988, J Exp Med 168, 1493-8; McGrogan et al., 1988, J Exp Med 168,2295-308). In SDS-PAGE, however, proMBP migrates as a smear of 50-90 kDathat is not visible in Coomassie-stained gels (Oxvig et al., 1993, JBiol Chem 268, 12243-6), probably due to its strong and unusualglycosylation (Oxvig et al., 1994, Biochem Mol Biol Int 33, 329-36;Oxvig et al., 1994, Biochim Biophys Acta 1201, 415-23). PAPP-A andproMBP are both produced in the placenta during pregnancy, but mainly indifferent cell types as shown by in situ hybridization (Bonno et al.,1994, Lab Invest 71, 560-6). Analyses by RT-PCR revealed that bothPAPP-A and proMBP mRNA are present in several reproductive andnonreproductive tissues, although the levels are lower than in theplacenta (Overgaard et al., 1999, Biol Reprod 61, 1083-9).

Clinical Use of PAPP-A

Clinically, depressed serum levels of PAPP-A are increasingly being usedas a predictor of Down's syndrome pregnancies (Brambati et al., 1993, BrJ Obstet Gynaecol 100, 324-6; Haddow et al., 1998, N Engl J Med 338,955-61; Wald et al., 1992, Bmj 305, 28; Wald et al., 1999, N Engl J Med341, 461-7), and it has been shown that PAPP-A serum levels are alsodepressed in other fetal abnormalities (Biagiotti et al., 1998, PrenatDiagn 18, 907-13; Spencer et al., 2000, Prenat Diagn 20, 411-6;Westergaard et al., 1983, Prenat Diagn 3, 225-32).

Further, the synthesis of PAPP-A in smooth muscle cells of the coronaryartery following angioplasty is increased (Bayes-Genis et al., 2000,Arterioscler Thromb Vasc Biol, in press), which is currently beingevaluated for potential clinical value. Data show that measurements ofproMBP in pregnancy serum also have a diagnostic value (Christiansen etal., 1999, Prenat Diagn 19, 905-10).

Proteolytic Activity of PAPP-A: Cleavage of IGFBP-4

Only recently, the putative metalloproteinase activity of PAPP-A hasbeen experimentally confirmed (Lawrence et al., 1999, Proc Natl Acad SciUSA 96, 3149-53). PAPP-A was partially purified from humanfibroblast-conditioned medium (HFCM) and shown to be responsible for theproteolytic activity of HFCM against insulin-like growth factor bindingprotein (IGFBP)-4. IGFBP's, of which six have been described, areimportant modulators of IGF-I and -II activity (Fowlkes, 1997, TrendsEndocrinol Metab 8, 299-306; Rajaram et al., 1997, Endocr Rev 18,801-31).

IGF-I and -II are essential polypeptides with potent anabolic andmitogenic actions both in vivo and in vitro. IGF bound to IGFBP-4 cannotinteract with its receptor, but bioactive IGF is released once thebinding protein is cleaved. Interestingly, cleavage of IGFBP-4 by PAPP-Astrictly requires the presence of IGF (Conover et al., 1993, J ClinInvest 91, 1129-37; Lawrence et al., 1999, Proc Natl Acad Sci USA 96,3149-53). PAPP-A secretion has also been demonstrated from osteoblastsand marrow stromal cells (Lawrence et al., 1999, Proc Natl Acad Sci USA96, 3149-53), from granulosa cells (Conover et al., 1999, J ClinEndocrinol Metab 84, 4742-5), and from vascular smooth muscle cells(Bayes-Genis et al., 2000, Arterioscler Thromb Vasc Biol, in press), allof which have known IGF-dependent IGFBP-4 proteinase activity.

IGFBP-5

Like IGFBP-4, IGFBP-5 cleavage has been widely reported to occur byunidentified proteinases is a number of tissues and conditioned media(Hwa et al., 1999, Endocr Rev 20, 761-87).

SUMMARY OF THE INVENTION

Pregnancy-Associated Plasma Protein-A2

The novel nucleic acid according to the invention has been isolated fromhuman placenta and characterised by means of sequencing analysis. Thenovel nucleotide sequence encodes a new polypeptide, PAPP-A2.

The amino acid sequence of PAPP-A2 is composed of a 233-residuepre-pro-piece and a 1558-residue mature portion. The mature portion ofPAPP-A2 is homologous with the mature portion of PAPP-A (approx. 45%identity), but the prepro-pieces do not show any similarity between thetwo proteins. Like PAPP-A, PAPP-A2 contains conserved amino acidstretches that classify it as a putative metalloproteinase of themetzincin superfamily.

PAPP-A2 has been expressed in a mammalian expression system, and it hasbeen demonstrated that PAPP-A2 is an active enzyme. Further, it has beenshown that PAPP-A2 cleaves IGFBP-5, Insulin Like Growth Factor BindingProtein 5. In comparison, the cleavage of IGFBP-4 by PAPP-A haspreviously been demonstrated.

A complementary DNA (cDNA) which encodes the full length form of PAPP-A2is identified, sequenced and isolated. The cDNA or portions of the cDNAis cloned into expression vectors for expression in a recombinant host.The cDNA is useful to produce recombinant full-length PAPP-A2 orfragments of PAPP-A2. The cDNA and the recombinant PAPP-A2 proteinderived therefrom are useful in the production of antibodies, diagnostickits, laboratory reagents and assays.

The cDNA and the recombinant PAPP-A2 protein may also be used toidentify compounds that affect PAPP-A2 function. PAPP-A2 antisenseoligonucleotides or antisense mimetics may be clinically useful forreducing the expression of PAPP-A2 protein and thereby antagonizing theeffects of PAPP-A. Similarly, the PAPP-A2 coding sequence can be usedfor gene therapy to introduce PAPP-A2 into target cells therebyenhancing the effects of PAPP-A2.

The invention furthermore pertains to PAPP-A2 for use as a therapeutictarget for the reduction or elimination of IGFBP-5 proteolytic activityin a cell.

It is furthermore an objective of the present invention to providemethods for use of PAPP-A2 for diagnostic purposes.

Other features and advantages of the invention will be apparent from thefollowing drawings and description hereof, from the following detaileddescription, and from the claims.

DEFINITIONS

As used herein, PAPP-A2 refers to an isolated PAPP-A2 polypeptide havingthe amino acid sequence listed in FIG. 1 (SEQ ID NO:2), or a variantthereof as defined herein. The PAPP-A2 according to the invention, or avariant thereof, may be produced by recombinant DNA technology, or thePAPP-A2 may be naturally occurring.

A PAPP-A2 encoding nucleotide sequence refers to an isolated nucleicacid having the sequence listed in FIG. 1 (SEQ ID NO:1), or a variantthereof as defined herein.

“Active” refers to those forms of PAPP-A2 which retain the biologicaland/or immunological activities of any naturally occurring PAPP-A2.

“Naturally occurring PAPP-A2” refers to PAPP-A2 produced by human cellsthat have not been genetically engineered and specifically contemplatesvarious PAPP-A2s arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation, acylation, or complexformation, covalent or noncovalent, with other polypeptides.

An “isolated polypeptide” is a protein, or a variant or fragmentthereof, which constitutes 90% or more of the protein contents of agiven preparation as evaluated by standard methods known in the art ofprotein chemistry.

“Derivative” refers to polypeptides derived from naturally occurringPAPP-A2 by chemical modifications such as ubiquitination, labeling(e.g., with radionuclides, various enzymes, etc.), pegylation(derivatization with polyethylene glycol), or by insertion (orsubstitution by chemical synthesis) of amino acids (amino acids) such asornithine, which do not normally occur in human proteins.

“Recombinant variant” refers to any polypeptide differing from naturallyoccurring PAPP-A2 by amino acid insertions, deletions, andsubstitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as proteolytic activityor cell adhesion, may be found e.g. by comparing parts of the sequenceof PAPP-A2 with structurally similar proteins (e.g. other metzincinfamily proteinases), with locally homologous proteins of known disulfidestructure, or by secondary structure predictions.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as, but not limited to, the replacement of aleucine with an isoleucine or valine, replacement of an aspartate with aglutamate, or replacement with a threonine with a serine, i.e.,conservative amino acid replacements. Further examples and definitionsfalling within the meaning of the term “substitutions” as applied hereinare provided in the detailed description of the invention herein below.

Amino acid “insertions” or “deletions” are typically in the range offrom about 1 amino acid to about 50 amino acids, such as from about 1amino acid to about 20 amino acids, for example from about 1 amino acidto about 20 amino acids, such as from about 1 amino acid to about 10amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a PAPP-A2 molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

Where desired, a “signal or leader sequence” can direct the polypeptide(full length PAPP-A2, or portions of the PAPP-A2 polypeptide) throughthe membrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

A polypeptide “fragment”, “portion”, or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, such as atleast about 17 or more amino acids in various embodiments. It may alsobe a longer stretch of residues up to intact PAPP-A2 in length. To beactive, any PAPP-A2 polypeptide or PAPP-A2 polypeptide fragment musthave sufficient length to display biologic and/or immunologic activityon their own, or when conjugated to a carrier protein such as keyholelimpet hemocyanin.

An “oligonucleotide” or polynucleotide “fragment”, “portion”, or“segment” is a stretch of the PAPP-A2 encoding sequence which is usefulin the expression of PAPP-A2 polypeptide fragments. It may also be astretch of nucleotide residues capable of being used in a polymerasechain reaction (PCR) or a hybridization procedure, typically foramplifying or revealing related parts of mRNA or DNA molecules. Inparticular, one or both oligonucleotide probes will comprise sequencethat is identical or complementary to a portion of PAPP-A2 where thereis little or no identity or complementarity with any known or prior artmolecule. For this purpose, such oligonucleotide probes will generallycomprise between about 10 nucleotides and 50 nucleotides, and preferablybetween about 15 nucleotides and about 30 nucleotides.

“Animal” as used herein may be defined to include human, domestic oragricultural (cats, dogs, cows, sheep, etc) or test species (mouse, rat,rabbit, etc).

“Recombinant” may also refer to a polynucleotide which encodes PAPP-A2and is prepared using recombinant DNA techniques. The DNAs which encodePAPP-A2 may also include allelic or recombinant variants and mutantsthereof.

“Nucleic acid probes” are prepared based on the cDNA sequences whichencode PAPP-A2 provided by the present invention. Nucleic acid probescomprise portions of the sequence having fewer nucleotides than about 6kb, usually fewer than about 1 kb. After appropriate testing toeliminate false positives, these probes may be used to determine whethermRNAs encoding PAPP-A2 are present in a cell or tissue or to isolatesimilar nucleic acid sequences from chromosomal DNA extracted from suchcells or tissues as described in (Walsh et al., 1992, PCR Methods Appl1, 241-50). Probes may be derived from naturally occurring orrecombinant single- or double-stranded nucleic acids or be chemicallysynthesized. They may be labeled by nick translation, Klenow fill-inreaction, PCR or other methods well known in the art. Probes of thepresent invention, their preparation and/or labeling are elaborated in(Sambrook et al., 1989); or (Ausubel et al., 1989).

Alternatively, recombinant variants encoding these PAPP-A2 or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide,including but not limited to activity, interchain affinities, orpolypeptide degradation or turnover rate. One example involves insertinga stop codon into the nucleotide sequence to limit the size of PAPP-A2so as to provide a molecule of smaller molecular weight.

“Expression vectors” are defined herein as DNA sequences that arerequired for the transcription of cloned copies of genes and thetranslation of their mRNAs in an appropriate host. Such vectors can beused to express eukaryotic genes in a variety of hosts such as bacteria,yeast, bluegreen algae, plant cells, insect cells and animal cells.

The term “antibody” as used herein includes both polyclonal andmonoclonal antibodies, as well as fragments thereof, such as, Fv, Faband F(ab)2 fragments that are capable of binding antigen or hapten. Itincludes conventional murine monoclonal antibodies as well as humanantibodies, and humanized forms of non-human antibodies, and it alsoincludes ‘antibodies’ isolated from phage antibody libraries.

“Ribozymes” are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by a endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that specifically and efficiently catalyze endonucleolyticcleavage of PAPP-A2 RNA sequences. Specific ribozyme cleavage siteswithin any potential RNA target are initially identified by scanning thetarget molecule for ribozyme cleavage sites which include the followingsequences, GUA, GUU and GUC. Once identified, short RNA sequences ofbetween fifteen (15) and twenty (20) ribonucleotides corresponding tothe region of the target gene containing the cleavage site may beevaluated for predicted structural features such as secondary structurethat may render the oligonucleotide sequence unsuitable. The suitabilityof candidate targets may also be evaluated by testing theiraccessibility to hybridization with complementary oligonucleotides,using ribonuclease protection assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the cDNA sequence (in 5′→3′ orientation) correspondingto the mRNA that encodes preproPAPP-A2. Only the coding part of thesequence and the terminal stop codon (*) is shown and is numbered1-5376. The translated polypeptide sequence of preproPAPP-A2 is alsoshown. The signal peptide cleavage site was predicted using SignalP V2.0to be after the alanine residue encoded by nt. 64-66 ((Nielsen et al.,1997, Protein Eng 10, 1-6), WWW prediction server is located atgenome.cbs.dtu.dk/). The signal peptide of preproPAPP-A2 (nt. 1-66, 22residues) is shown in bold. The nucleotide sequence of this figurerepresents nt. 1 to 5376 of SEQ ID NO:1. The protein sequence of thisfigure is illustrated as SEQ ID NO:2.

FIG. 2 is a schematic drawing of the relationship between PAPP-A(Kristensen et al., 1994, Biochemistry 33, 1592-8), and sequencestretches contained within two genomic clones with homology to theN-terminal end (hom-N, coding portion of accession number AL031734) andthe C-terminal end (hom-C, coding portion of accession number AL031290)of PAPP-A, when translated into amino acid sequence. This figure alsoillustrates the method by which a cDNA sequence with homology to themidregion of PAPP-A was obtained. Hom-N, hom-C, and the midregiontogether encodes the complete sequence of a novel protein, PAPP-A2,which is a homolog of PAPP-A. The midregion was obtained by PCR usingspecifically primed (primer RT-N-mid), reversed transcribed humanplacental mRNA as the template, and primers PR-mid5 and PR-mid3 for thePCR (Table 1). To obtain a cDNA construct encoding the full-lengthPAPP-A2, cDNA clones corresponding to the genomic clones hom-N and hom-Cwere also obtained using cDNA synthesized with specifically primedplacental mRNA as the template (primers not shown, see Table 1). Thisrequired identification of a signal peptide stretch (in hom-N) and astop codon (at the 3′ end of hom-C), as detailed in the main text. Allprimers used are shown in Table 1. Note: The relative positions of thesequences depicted here are in accordance with the experimentsperformed, but the figure is not accurately drawn to scale.

FIGS. 3A-3G show the amino acid sequence of preproPAPP-A2 (SEQ ID NO:2)aligned with preproPAPP-A. The deduced amino acid sequence ofpreproPAPP-A2 (PA2) was aligned with the sequence of preproPAPP-A (PA)((Haaning et al., 1996, Eur J Biochem 237, 159-63), AAC50543) usingCLUSTAL W (Thompson et al., 1994, Nucleic Acids Res 22, 4673-80).Because the prepro-portion of PAPP-A did not show significant identitywith the corresponding region of PAPP-A2, the alignment was manuallyadjusted to emphasize difference in length of pro-peptides. Arrowsindicate the N-termini of the mature proteins as found earlier forPAPP-A (Kristensen et al., 1994, Biochemistry 33, 1592-8) (Glu-81), andhere for PAPP-A2 (Ser-234). Putative signal peptides, strongly predictedusing SignalP V2.0 (Nielsen et al., 1997, Protein Eng 10, 1-6) are shownwith lower case letters. The pro-portion of PAPP-A2 contains one othercandidate initiation codon corresponding to Met-168, but no signalpeptide was predicted following this residue using SignalP. The sequencemotifs of PAPP-A (Kristensen et al., 1994, Biochemistry 33, 1592-8) arealso found in PAPP-A2: The catalytic zinc binding motif and residues ofthe putative Met-turn are underlined and bolded in both sequences.Lin-notch motifs (LNR1-3) and short consensus repeats (SCR-1-5) areboxed. All cysteines of mature PAPP-A are also found in PAPP-A2. Inaddition, the secreted form of PAPP-A2 has four cysteine residues(Cys-343, Cys-533, Cys-618, and Cys-1268) with no counterpart in PAPP-A.

FIG. 4 shows PAPP-A2 by Western blotting and Coomassie staining. Mediumfrom transfected 293T cells was Western blotted using monoclonalanti-c-myc. Lane 1, cells transfected with empty vector; lane 2, cellstransfected with cDNA encoding wild-type PAPP-A2 C-terminally taggedwith the c-myc peptide (pPA2-mH), non-reduced; lane 3, cells transfectedwith or cDNA encoding PAPP-A2 with an inactivating E734Q mutation(pPA2-KO-mH), non-reduced; lane 4, as lane 2, but reduced. RecombinantPAPP-A2 was purified by nickel affinity chromatography from serum freemedium of cells transfected with pPA2-KO-mH, to eliminate possibleautocatalysis (lane 5, reduced).

FIG. 5 shows the activity of PAPP-A2 against IGFBP-1-6. Medium from 293Tcells transfected with empty vector (−), or cDNA encoding PAPP-A2 (pPA2)(+) was incubated with each of the six IGFBPs (BP1-BP6), and theactivity was assessed by ligand blotting using radiolabeled IGF-II.Complete cleavage of IGFBP-5 is evident from the absence of a signal inthe BP5+ lane. Partial degradation of IGFBP-3 is also evident.

FIG. 6 shows proteolytic activity of PAPP-A2 against IGFBP-5. Mediumfrom 293T cells transfected with empty vector (lane 1), cDNA encodingPAPP-A2 with an inactivating E734Q mutation (pPA2-KO) (lane 2), or cDNAencoding wild-type PAPP-A2 (pPA2) (lanes 3-6) was incubated withC-terminally c-myc tagged rIGFBP-5. Proteolytic activity was assessed byWestern blotting using anti-c-myc. ‘i’ denotes intact rIGFBP-5; ‘c’denotes the detectable C-terminal c-myc tagged cleavage product. In theabsence of inhibitors, wild-type PAPP-A2 degraded all rIGFBP-5 (lane 3).The PAPP-A2 activity was abolished by 10 mM phenantroline (lane 4) and 5mM EDTA (lane 5), but not affected by 100 μM 3,4-DCI (lane 6).Coomassie-stained SDS-PAGE of purified rIGFBP-5 is shown before (lane 7)and after (lane 8) digestion with purified PAPP-A2. A Western blot ofthe same digest, using anti-c-myc, is also shown (lane 9). Sequenceanalysis revealed that PAPP-A2 cleaves IGFBP-5 at one site, betweenSer-142 and Lys-143.

FIGS. 7A-7C show the cDNA sequence of the PAPP-A2 mRNA coding regiondirectly followed by the sequence of the 3′UTR. The sequence of the3′UTR was obtained as detailed in Example 6.3 The first 5376 nucleotidesof this sequence (nt. 1-5376) represents the coding sequence asillustrated in FIG. 1 and SEQ ID NO:1 (nt. 1-5376). Nucleotides5377-8527 of this sequence corresponds to the 3′UTR of the PAPP-A2 mRNAas illustrated in SEQ ID NO:3 (nt. 5377-8527).

FIG. 8 shows the disulfide structure of the PAPP-A subunit in thePAPP-A/proMBP complex (upper bar). Cysteine containing peptidesoriginating from the PAPP-A/proMBP complex were isolated by degradingPAPP-A/proMBP complex with proteinases and cyanogen bromide followed bystandard HPLC. Peptides were identified by amino acid analysis,N-terminal sequence analysis, and by mass spectrometry (Overgaard, M.T., Oxvig, C., unpublished). Disulfide bonds are shown by thin lines.Two cysteine residues form inter-chain disulfide bridges to proMBP, andone forms an inter-chain bridge to PAPP-A causing it to be a dimer (asindicated). Asterisks mark a cysteine residue to which no partner hasbeen found. The cysteine residues present in mature PAPP-A is alsopresent in mature PAPP-A2 (see FIG. 3). It is reasonable to assume thatthe disulfide pairing of PAPP-A2 is the same. Thus, this information isvaluable in determination of boundary regions for expression of isolateddomains (fragments) of PAPP-A2. The gene structure of PAPP-A is alsoshow (lower bar). Exon/intron boundaries are based on comparison ofPAPP-A cDNA (AN X68280) with genomic sequences (ANs AB020878, AL353141,and AL137024). The central bar shows putative domains of PAPP-A based oninformation of the upper and lower bars.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of a Nucleotide Sequence Encoding PAPP-A2

The present invention in one aspect relates to a novel cDNA sequenceencoding a protein with global homology to pregnancy-associated plasmaprotein-A (PAPP-A). This protein has been denoted PAPP-A2. The completenucleotide sequence of PAPP-A2 has been obtained from mRNA isolated fromhuman placenta (Example 1). The complete nucleotide sequence (SEQ IDNO:1) and translated amino acid sequence (SEQ ID NO:2) of PAPP-A2 areboth shown in FIG. 1.

Homology of PAPP-A2 with PAPP-A is evident upon alignment of the twoamino acid sequences as shown in FIG. 3. PAPP-A2 and PAPP-A shareapproximately 45% of their amino acid residues. Sequence motifs known tobe important for the function of PAPP-A (Kristensen et al., 1994,Biochemistry 33, 1592-8; Lawrence et al., 1999, Proc Natl Acad Sci USA96, 3149-53; Overgaard et al., 2000, J Biol Chem) are also found inPAPP-A2. Principally, PAPP-A2 contains an elongated zinc binding motif(HEXXHXXGXXH (SEQ ID NO:3), amino acids shown by one letter code) atposition 733-743 (FIG. 2). This motif and a structurally importantmethionine residue, are strictly conserved within the metzincins, asuperfamily of zinc peptidases (Bode et al., 1993, FEBS Lett 331,134-40; Stocker et al., 1995, Protein Sci 4, 823-40).

Like PAPP-A, PAPP-A2 is synthesized as a prepro-protein. PreproPAPP-A2has 1791 amino acids (FIG. 1). There is no homology between theprepro-portions of PAPP-A and PAPP-A2. Further, the prepro-portions ofthe two proteins differ significantly in length. The PAPP-A2prepro-peptide has 233 residues (FIG. 3); the PAPP-A prepro-peptide has80 residues (Haaning et al., 1996, Eur J Biochem 237, 159-63).

Uses of the Nucleotide Sequence Encoding PAPP-A2

The nucleotide sequence encoding PAPP-A2 (or its complement) havenumerous applications in techniques known to those skilled in the art ofmolecular biology. These techniques include use as hybridization probes,use in the construction of oligomers for PCR, use in the recombinantproduction of PAPP-A2 or fragments hereof, and use in generation ofanti-sense DNA or RNA, their chemical analogs (e.g. PNA or LNA) and thelike. Uses of nucleotides encoding PAPP-A2 disclosed herein areexemplary of known techniques and are not intended to limit their use inany technique known to a person of ordinary skill in the art.Furthermore, the nucleotide sequences disclosed herein may be used inmolecular biology techniques that have not yet been developed, providedthe new techniques rely on properties of nucleotide sequences that arecurrently known, e.g., the triplet genetic code, specific base pairinteractions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of PAPP-A2-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequence of any known and naturally occurring gene may be produced. Theinvention has specifically contemplated each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring PAPP-A2, and all suchvariations are to be considered as being specifically disclosed.

Although the nucleotide sequences which encode PAPP-A2 and/or itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring PAPP-A2 under stringent conditions,it may be advantageous to produce nucleotide sequences encoding PAPP-A2or its derivatives possessing a substantially different codon usage.Codons can be selected to increase the rate at which expression of thepeptide occurs in a particular prokaryotic or eukaryotic expression hostin accordance with the frequency with which particular codons areutilized by the host. Other reasons for substantially altering thenucleotide sequence encoding PAPP-A2 and/or its derivatives withoutaltering the encoded amino acid sequence include the production of RNAtranscripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

Nucleotide sequences encoding PAPP-A2 may be joined to a variety ofother nucleotide sequences by means of well established recombinant DNAtechniques (Sambrook et al., 1989). Useful nucleotide sequences forjoining to PAPP-A2 include an assortment of cloning vectors, e.g.,plasmids, cosmids, lambda phage derivatives, phagemids, and the like,that are well known in the art. Vectors of interest include expressionvectors, replication vectors, probe generation vectors, sequencingvectors, and the like. In general, vectors of interest may contain anorigin of replication functional in at least one organism, convenientrestriction endonuclease sensitive sites, and selectable markers for thehost cell.

Another aspect of the subject invention is to provide forPAPP-A2-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences encodingPAPP-A2. Such probes may also be used for the detection of similarPAPP-A2 encoding sequences and should preferably contain at least 50% ofthe nucleotides from the conserved region or active site. Thehybridization probes of the subject invention may be derived from thenucleotide sequences of the SEQ ID NO:1 or from genomic sequencesincluding promoters, enhancer elements and/or possible introns of therespective naturally occurring PAPP-A2. Hybridization probes may belabeled by a variety of reporter groups, including radionuclides such as32P or 35S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

PCR as described (U.S. Pat. Nos. 4,683,195; and 4,965,188) providesadditional uses for oligonucleotides based upon the nucleotide sequencewhich encodes PAPP-A2. Such probes used in PCR may be of recombinantorigin, may be chemically synthesized, or a mixture of both and comprisea discrete nucleotide sequence for diagnostic use or a degenerate poolof possible sequences for identification of closely related genomicsequences.

Other means of producing specific hybridization probes for PAPP-A2 DNAsinclude the cloning of nucleic acid sequences encoding PAPP-A2 orPAPP-A2 derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art and are commercially available and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate radioactively labeled nucleotides.

It is possible to produce a DNA sequence, or portions thereof, encodingPAPP-A2 and their derivatives entirely by synthetic chemistry, afterwhich the gene can be inserted into any of the many available DNAvectors using reagents, vectors and cells that are known in the art atthe time of the filing of this application. Moreover, syntheticchemistry may be used to introduce mutations into the PAPP-A2 sequencesor any portion thereof.

The nucleotide sequence can be used in an assay to detect diseaseassociated with abnormal levels of expression of PAPP-A2. The nucleotidesequence can be labeled by methods known in the art and added to a fluidor tissue sample from a patient under hybridizing conditions. After anincubation period, the sample is washed with a compatible fluid whichoptionally contains a dye (or other label requiring a developer) if thenucleotide has been labeled with an enzyme. After the compatible fluidis rinsed off, the dye is quantitated and compared with a standard.Alternatively, levels of PAPP-A2 mRNA can be measured by micro arraytechniques using immobilized probes. Expression in samples can also beevaluated by (semi-quantitative) RT-PCR. Expression in samples canalternatively be evaluated by techniques based on hybridization. Forexample, in situ hybridization can be used to detect PAPP-A2 mRNA. Thistechnique has the advantage that it locates the cells that synthesizethe mRNA, but also is less sensitive than RT-PCR.

Included in the scope of the invention are oligoribonucleotidesequences, that include antisense RNA and DNA molecules and ribozymesthat function to inhibit translation of PAPP-A2. Antisense techniquesare known in the art and may be applied herein. Both antisense RNA andDNA molecules and ribozymes of the invention may be prepared by anymethod known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligodeoxyribonucleotideswell known in the art such as for example solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding the antisenseRNA molecule. Such DNA sequences may be incorporated into a wide varietyof vectors which incorporate suitable RNA polymerase promoters such asthe T7 or SP6 polymerase promoters. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly,depending on the promoter used, can be introduced stably into celllines.

The invention also relates to unknown PAPP-A2 genes isolated from otherspecies and alleles of the PAPP-A2 gene, in which PAPP-A2 orthologues orhomologues exists. A bacteriophage cDNA library may be screened, underconditions of reduced stringency, using a radioactively labeled fragmentof the human PAPP-A2 clone described herein. Alternatively the humanPAPP-A2 sequence can be used to design degenerate or fully degenerateoligonucleotide probes which can be used as PCR probes or to screenbacteriophage cDNA libraries. The PCR product may be subcloned andsequenced to insure that the amplified sequences represent the PAPP-A2sequences. The PCR fragment may be used to isolate a full length PAPP-A2clone by radioactively labeling the amplified fragment and screening abacteriophage cDNA library. Alternatively, the labeled fragment may beused to screen a genomic library. For a review of cloning strategieswhich may be used, see e.g., (Ausubel et al., 1989; Sambrook et al.,1989).

Expression of Recombinant PAPP-A2

In order to express a biologically active proteinase, the nucleotidesequence coding for the protein, or a functional equivalent, can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. For example, recombinant protein can beused for immunization to obtain antibodies, as a laboratory reagent, andin diagnostic kits.

More specifically, methods which are well known to those skilled in theart can be used to construct expression vectors containing the PAPP-A2sequence and appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See e.g.,the techniques described in (Ausubel et al., 1989; Sambrook et al.,1989).

Further, expression vectors containing fragments of the PAPP-A2 encodingsequence may also be constructed. In particular, this may be relevantfor the use of portions of the PAPP-A2 polypeptide as an antigen forimmunization. In addition, the coding sequence of PAPP-A2 or fragmentshereof may be cloned in frame with a coding nucleotide sequence presentin the vector to result in a fusion protein or a ‘tagged’ PAPP-A2protein. For example, such a fusion protein may be composed of PAPP-A2and GST, and such tag may be a c-myc tag (for detection) and/or ahistidine tag (for purification).

A variety of host-expression vector systems may be utilized to expressthe PAPP-A2 coding sequence or fragments hereof. These include but arenot limited to microorganisms such as bacteria transformed withrecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing the PAPP-A2 coding sequence; yeast transformed withrecombinant yeast expression vectors containing the PAPP-A2 codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the PAPP-A2 coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the PAPP-A2 coding sequence; or animal cell systemsinfected with recombinant virus expression vectors (e.g., adenovirus,vaccinia virus, human tumor cells) including cell lines engineered tocontain multiple copies of the PAPP-A2 DNA either stably amplified(CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g.,murine cell lines).

The expression elements of these systems vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theCMV promotor, the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) may be used; when generating cell lines that contain multiplecopies of the PAPP-A2 DNA SV40-, BPV- and EBV-based vectors may be usedwith an appropriate selectable marker.

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, infection, protoplast fusion, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce PAPP-A2 protein.Identification of PAPP-A2 expressing host cell clones may be done byseveral means, including but not limited to immunological reactivitywith anti-PAPP-A2 antibodies, and the presence of host cell-associatedPAPP-A2 activity.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the PAPP-A2expressed. For example, when large quantities of PAPP-A2 are to beproduced, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther and Muller-Hill, 1983, Embo J 2, 1791-4), in which thePAPP-A2 coding sequence may be ligated into the vector in frame with thelac Z coding region so that a hybrid AS-lac Z protein is produced. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety. In yeast, anumber of vectors containing constitutive or inducible promoters may beused. For a review, see (Ausubel et al., 1989; Bitter et al., 1987,Methods Enzymol 153, 516-44; Rosenfeld, 1999, Methods Enzymol 306,154-69).

In cases where plant expression vectors are used, the expression of thePAPP-A2 coding sequence may be driven by any of a number of promoters.For example, viral promoters such as the 35S RNA and 19S RNA promotersof CaMV may be used (Gmunder and Kohli, 1989, Mol Gen Genet 220,95-101); alternatively, plant promoters such as the small subunit ofRUBISCO (Broglie et al., 1984, Science 224, 838-43).

An alternative expression system which could be used to express PAPP-A2is an insect system. In one such system, Baculovirus is used as a vectorto express foreign genes. The virus then grows in the insect cells. ThePAPP-A2 coding sequence may be cloned into non-essential regions (forexample the polyhedron gene) of the virus and placed under control of aBaculovirus promoter. These recombinant viruses are then used to infectinsect cells in which the inserted gene is expressed. For example, see(Smith et al., 1983, Mol Cell Biol 3, 2156-65).

A variety of mammalian expression vectors may be used to expressrecombinant PAPP-A2 in mammalian cells. Commercially-available mammalianexpression vectors which may be suitable for recombinant PAPP-A2expression, include but are not limited to, pMC1neo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2)(ATCC 37110), pcDNA3.1 and its derivatives (Stratagene). Cell linesderived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, CV-1, COS-1,COS-7, CHO-K1, 3T3, NIH3T3, HeLa, C127I, BS-C-1, MRC-5, and 293.Further, in mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the PAPP-A2 coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing PAPP-A2 in infected hosts. See for example (Logan and Shenk,1984, Proc Natl Acad Sci USA 81, 3655-9). Alternatively, the vaccinia7.5K promoter may be used. See for example (Mackett et al., 1982, ProcNatl Acad Sci USA 79, 7415-9).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressPAPP-A2 may be engineered. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withPAPP-A2 DNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines.

Some applications of the recombinant PAPP-A2 may require the protein tobe in purified or partially purified form. Recombinantly expressedPAPP-A2 or fragments of the PAPP-A2 polypeptide can be isolated byliquid chromatography. Various methods of protein purification wellknown in the art include those described in for example (Scopes, 1987).Alternatively, recombinant PAPP-A2 fusion proteins or ‘tagged’ PAPP-A2may be purified by affinity chromatography. Further, antibodies raisedagainst PAPP-A2 may be used for purification by immunoaffinitychromatography.

Recombinant variant of PAPP-A2 may be produced by site directedmutagenesis. In some applications of PAPP-A2 such variants may bepreferred due to for example increased protein stability, or changes inactivity.

Production and Uses of Antibodies Against PAPP-A2

The recombinant protein may be used to generate antibodies. Monospecificantibodies to PAPP-A2 can be purified from mammalian antisera containingantibodies reactive against PAPP-A2 or can be prepared as monoclonalantibodies reactive with PAPP-A2 using standard techniques.

Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for PAPP-A2. Homogenous binding as used herein refers tothe ability of the antibody species to bind to a specific antigen orepitope, such as those associated with the PAPP-A2, as described above.PAPP-A2 specific antibodies are raised by immunizing animals such asmice, rats, guinea pigs, rabbits, goats, horses and the like, withrabbits or mice being preferred, with an appropriate concentration ofPAPP-A2 either with or without an immune adjuvant. For example,antibodies specific against PAPP-A2 can be used for the purification ofnative and recombinant PAPP-A2, as a laboratory reagent, and in antibodybased diagnostic kits.

Monoclonal antibodies (mAb) reactive with PAPP-A2 can be prepared byconventional methods, such as by immunizing inbred mice with PAPP-A2.The mice are immunized with about 0.1 mg to about 10 mg, preferablyabout 1 mg, of PAPP-A2 in about 0.5 ml buffer or saline incorporated inan equal volume of an acceptable adjuvant. Freund's complete adjuvant ispreferred. The mice receive an initial immunization on day 0 and arerested for about 3 to about 30 weeks. Immunized mice are given one ormore booster immunizations of about 0.1 to about 10 mg of PAPP-A2 in abuffer solution such as phosphate buffered saline (PBS) by theintravenous (IV) route. Lymphocytes from antibody-positive mice areobtained by removing spleens from immunized mice by standard proceduresknown in the art. Hybridoma cells are produced by mixing the spleniclymphocytes with an appropriate fusion partner under conditions whichwill allow the formation of stable hybridomas. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected form growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using PAPP-A2 as the antigen. The culture fluids are also testedin the Ouchterlony precipitation assay to determine the isotype of themAb. Hybridoma cells from antibody positive wells are then cloned. Fordetails, see (Peters and Baumgarten, 1992).

In vitro production of anti-PAPP-A2 is carried out by growing thehydridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique (Crowther, 1995).

The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in (Clackson et al., 1991,Nature 352, 624-8; Marks et al., 1991, J Mol Biol 222, 581-97), forexample. Identified phage antibodies can be produced by expression inbacteria.

Methods such as those described above may be used to producemonospecific antibodies specific for PAPP-A2 polypeptide fragments orfull-length nascent PAPP-A2 polypeptide.

PAPP-A2 antibody affinity columns can be made by adding the antibodiesto a gel support, such as Affigel-10 (Biorad), a gel support which ispre-activated with N-hydroxysuccinimide esters such that the antibodiesform covalent linkages with the agarose gel bead support. The antibodiesare then coupled to the gel via amide bonds with the spacer arm. Theremaining activated esters are then quenched with 1M ethanolamine HCl(pH 8). The column is washed with water followed by 0.23 M glycine HCl(pH 2.6) to remove any non-conjugated antibody or extraneous protein.The column is then equilibrated in phosphate buffered saline (pH 7.3)and the cell culture supernatants or cell extracts containing PAPP-A2 orPAPP-A2 fragments are slowly passed through the column. The column isthen washed, and the protein is eluted. The purified PAPP-A2 protein isthen dialyzed against phosphate buffered saline.

Native PAPP-A2 from sources such as human plasma or serum, tissueextracts, or media from nontransfected cell lines (that endogenouslysecrete PAPP-A2) may also be purified by use of an antibody affinitycolumn.

Using polyclonal or monoclonal antibodies against PAPP-A2 a number ofassays may be constructed for measurement of PAPP-A2 antigen in bodyfluids or tissue and cell extracts. Kits based on antibodies may be usedfor diagnostic purposes. The assays include, but are not limited to,precipitation, passive agglutination, enzyme-linked immunosorbent assay(ELISA) techniques, and radioimmunoassay (RIA) techniques.

For example, in one such ELISA, a sandwich assay can be constructedwhere antigen present in an sample is caught by immobilized polyclonalanti(PAPP-A2). Detection is then performed by the use of one or moremonoclonal PAPP-A2 antibodies and peroxidase conjugated anti(murineIgG). In another assay, antigen present in an sample is caught byimmobilized polyclonal anti(PAPP-A2), and detected using biotinylatedpolyclonal anti(PAPP-A2). For further examples and details, see(Crowther, 1995). Assays can be calibrated using purified PAPP-A2 toconstruct a standard curve by serial dilution. The concentration ofPAPP-A2 in solution in a purified form can be accurately measured byamino acid analysis (Sottrup-Jensen, 1993, Biochem Mol Biol Int 30,789-94).

Polyclonal antibodies may be used to inhibit the biological activity ofPAPP-A2. Specifically, in analogy with the inhibition of the IGFBP-4proteolytic activity of PAPP-A by polyclonal PAPP-A antibodies (Lawrenceet al., 1999, Proc Natl Acad Sci USA 96, 3149-53), anti(PAPP-A2) may beused to inhibit the proteolytic activity of PAPP-A2. Certain monoclonalantibodies may also be inhibitory towards the activity of PAPP-A2. Suchmonoclonal antibodies are likely to recognize an epitope in closeproximity to the active site of PAPP-A2, but the inhibitory activity mayalso be based on binding to epitopes other than those close to theactive site. Inhibitory monoclonal antibodies can be obtained byimmunization with PAPP-A2, PAPP-A2 fragments, with peptides derived fromPAPP-A2.

Inhibitory (monoclonal) antibodies may have therapeutic value inconditions of pathologies in which it may be desirable to decrease theactivity of PAPP-A2.

Activity of PAPP-A2

Like PAPP-A, PAPP-A2 contains conserved amino acid stretches thatclassify it as a putative metalloproteinase of the metzincin superfamily(Stocker et al., 1995, Protein Sci 4, 823-40). It has beenexperimentally verified that PAPP-A2 does exhibit proteolytic activityby demonstrating its cleavage of insulin-like growth factor bindingprotein (IGFBP)-5 (Example 6.7).

In general, proteolytic activity of PAPP-A2 against potential proteinsubstrates may be evaluated by the incubation of purified or partiallypurified PAPP-A2 with the potential substrate under a variety ofexperimental conditions (such as for example temperature, buffercomposition, ionic strength, and pH). Enzymatic activity of PAPP-A2against the protein in question can be evaluated by SDS-PAGE (in whichdegradation or release of well defined proteolytic fragment(s) will beevident), or by high-pressure liquid chromatographic detection ofreleased peptide(s). By means of such procedures, other substratetargets of PAPP-A2 may be identified. Incubation with a variant ofPAPP-A2 where, for example, a residue in the active site has beensubstituted to obtain an inactive enzyme, serves as a proper negativecontrol.

Random peptide libraries consisting of all possible combinations ofamino acids attached to a solid phase support may be used to identifypeptides that can be cleaved by PAPP-A2. Identification of such peptidesmay be accomplished by screening a peptide library with recombinantsoluble PAPP-A2. Methods for expression and purification of the enzymeare described above and may be used to express recombinant full lengthPAPP-A2 or fragments, analogs, or derivatives thereof depending on thefunctional domains of interest. For further details, see (Meldal, 1998,Methods Mol Biol 87, 65-74; Meldal, 1998, Methods Mol Biol 87, 51-7).Alternatively, peptide substrates may be derived from identified proteinsubstrates of PAPP-A2.

Alternatively, phage display of peptide libraries may be used toidentify peptides that can be cleaved by PAPP-A2 (Matthews and Wells,1993, Science 260, 1113-7).

Peptides that function as PAPP-A2 substrates may function in assays forthe detection of PAPP-A2 proteolytic activity in body fluids or tissueand cell extracts. Substrate peptides may be derivatized to function inan assay based on quenched-fluorescence (Meldal, 1998, Methods Mol Biol87, 65-74). Kits based on such, or other, techniques may be used fordiagnostic purposes in pathologies where measurement of PAPP-A2 activityis relevant.

Identification of Agents that Modify the Activity of PAPP-A2

An assay for the detection of PAPP-A2 proteolytic activity, as describedabove, provides a method for the identification of molecules that modifythe activity of PAPP-A2. Such molecules may be, for example, peptides,derivatized peptides, hydroxamic acid derivatized peptides, smallorganic molecules, or antibodies.

The screening of peptide libraries can be used to discoverpharmaceutical agents that act to modulate and/or inhibit the biologicalactivity of PAPP-A2. Methods for expression and purification of theenzyme are described above and may be used to express recombinant fulllength PAPP-A2 or fragments, analogs, or derivatives thereof dependingon the functional domains of interest. Random peptide librariesconsisting of all possible combinations of amino acids attached to asolid phase support may be used to identify peptides that are able tomodulate and/or inhibit PAPP-A2 activity by binding to the active siteor other sites of PAPP-A2. For example, see (Meldal, 1998, Methods MolBiol 87, 75-82).

Similarly, combinatorial chemistry may be used to identify low molecularweight organic molecules that affect the activity of PAPP-A2.

Measurement of Complexes of PAPP-A or PAPP-A2

PAPP-A primarily exists in pregnancy serum as a disulfide bound 2:2complex with the proform of eosinophil major basic protein (proMBP),PAPP-A/proMBP. In addition to the PAPP-A/proMBP complex, proMBP existsin the circulation as a disulfide bound 2:2 complex with angiotensin(ANG), proMBP/ANG, and a fraction of this complex is further complexedto a fragment of complement component C3dg (PROMBP/ANG/C3dg) (Oxvig,1995; Christiansen, 2000).

The level of complexes comprising PAPP-A and/or PAPP-A2 and/or proMBP inbody fluids of an individual may be indicative of predisposition to aclinical condition or indicative of the presense of a clinicalcondition. Accordingly, the present invention in one embodiment isdirected towards a method of diagnosing a clinical condition ordiagnosing predisposition to said clinical condition in an individualcomprising the steps of

-   -   a) providing a body sample from said individual; and    -   b) measuring the level of a complex selected from the group        consisting of PAPP-A/proMBP, PAPP-A2/proMBP, PAPP-A/PAPP-A2,        PAPP-A/PAPP-A2/proMBP, proMBP/ANG and proMBP/ANG/C3dg in said        body fluid sample; and    -   c) diagnosing the clinical condition or diagnosing        predisposition to the clinical condition, wherein the level of        the complex above or below a predetermined value is indicative        of the clinical condition or predisposition to the clinical        condition.

Furthermore, the levels of complexes comprising PAPP-A and/or PAPP-A2and/or proMBP in body fluids of a mammalian mother may be indicative ofpredisposition to a clinical condition or indicative of the presense ofa clinical condition in a fetus of said mother. Hence, the presentinvention provides methods of diagnosing a clinical condition ordiagnosing predisposition to said clinical condition in a mammalianfetus comprising the steps of

-   -   a) providing a body fluid sample from the mother of said fetus;        and    -   b) measuring the level of a complex selected from the group        consisting of PAPP-A/proMBP, PAPP-A2/proMBP, PAPP-A/PAPP-A2,        PAPP-A/PAPP-A2/proMBP, proMBP/ANG and proMBP/ANG/C3dg in said        body fluid sample; and    -   c) diagnosing the clinical condition or diagnosing        predisposition to the clinical condition, wherein the level of        the complex above or below a predetermined value is indicative        of the clinical condition or predisposition to the clinical        condition.

In particular, according to the present method the level of one or moreof the following complexes may be determined:

PAPP-A/proMBP

PAPP-A2 and proMBP (PAPP-A2/proMBP)

PAPP-A2 and PAPP-A (PAPP-A/PAPP-A2)

PAPP-A/PAPP-A2 with proMBP (PAPP-A/PAPP-A/proMBP)

proMBP/ANG

proMBP/ANG/C3dg

The level of complexes comprising PAPP-A and/or PAPP-A2 and/or proMBP ina body fluid sample may be determined by any conventional method knownto the person skilled in the art. For example, the level can be measuredby a method comprising the use of immunospecific reagents specificallyinteracting with one or more components of the complex desirable tomeasure, such as immunospecific reagents specifically interacting withPAPP-A, PAPP-A2, proMBP, ANG or C3gd. Immunospecific reagents may forexample bemonoclonal antibodies, polyclonal antibodies and/or antigenbinding fragments thereof, specific towards the individual components ofthe complex.

Such methods include but are not limited to sandwich ELISA, wherein animmunospecific reagent specifically recognising one component of thecomplex is employed as catching antibody and another immunospecificreagent specifically recognising another component if the complex isemployed as detection antibody. The detection antibody is preferablyeither directly or indirectly detectable, for example the detectionantibody may be directly coupled to a detectable label or the detectionantibody may be capable of interacting with another agent which iscoupled to a detectable label.

A detectable label may for example be a fluorescent label, achromatophore, a radioactive label, a heavy metal or an enzyme.

For example, the level of PAPP-A/proMBP complexes in a body fluid samplemay be determined by sandwich ELISA using a PAPP-A specific monoclonalor polyclonal antibody for catching and a proMBP specific monoclonal orpolyclonal antibody for detection or the level of proMBP/ANG in a bodyfluid sample may be determined by sandwich ELISA using a proMBP specificmonoclonal or polyclonal antibody for catching and a ANG specificmonoclonal or polyclonal antibody for detection.

The clinical condition may be any clinical condition which may bediagnosed by the level of complexes comprising PAPP-A and/or PAPP-A2and/or proMBP or wherein predisposition may be diagnosed by the level ofcomplexes comprising PAPP-A and/or PAPP-A2 and/or proMBP. The clinicalcondition may for example be selected from the group comprising Down'ssyndrome, preeclampsia and acute coronary syndrome, including unstableangina and myocardial infarction.

The body fluid sample may be any useful body fluid sample, such as ablood sample including a serum sample, a urine sample, a saliva sampleor an amniotic fluid sample.

In particular, the level of PAPP-A/proMBP may be determined when theclinical condition is selected from the group consisting of Down'ssyndrome, and acute coronary syndrome including unstable angina andmyocardial infarction.

In one embodiment of the present invention diagnosing Down's syndrome ordiagnosing predisposition to Down's syndrome, comprises determining thelevel of PAPP-A/proMBP, wherein the level of PAPP-A/proMBP below apredetermined value is indicative of the Down's syndrome orpredisposition to Down's syndrome.

In another embodiment of the present invention diagnosing acute coronarysyndrome, including unstable angina and myocardial infarction ordiagnosing predisposition to acute coronary syndrome, including unstableangina and myocardial infarction, comprises determining the level ofPAPP-A/proMBP, wherein the level of PAPP-A/proMBP above a predeterminedvalue is indicative of the acute coronary syndrome, including unstableangina and myocardial infarction or predisposition to acute coronarysyndrome, including unstable angina and myocardial infarction.

In yet another embodiment the level of proMBP/ANG may be determined todiagnose predisposition to Down's syndrome or to diagnose Down'ssyndrome. All the above mentioned methods of diagnosis may also beperformed in combination with one or more other methods of diagnosis. Inaddition, more than one different diagnosis according to the presentinvention may be performed, for example it is possible to measure thelevel of more than one complex or to measure the level of one complex indifferent body samples.

Use of PAPP-A2 to Generate Natural Proteolytic Fragments

PAPP-A2 may be used to generate natural fragments of proteins that arespecifically cleaved by PAPP-A2. As in the case of IGFBP-5 (see Examples6.7 and 6.9), such fragments may have biological effects different fromintact IGFBP-5. Fragments can be purified by standard chromatographyafter cleavage with purified PAPP-A2 (see Example 6.9).

Design of Fragments of PAPP-A2 for Expression

Because all cysteine residues found in mature PAPP-A are also found inmature PAPP-A2 (see FIG. 3), the pattern of disulfide bonds can beassumed to be the same for PAPP-A2 for those common cysteine residues.Therefore, knowledge of the disulfide structure of the PAPP-A subunit(see FIG. 8) can be used to rationally design fragments of PAPP-A2 inwhich pairing of all cysteine residues is possible. Putative domainboundaries of PAPP-A2 can be defined based on the disulfide structureshown in FIG. 8. Those domains can be expressed separately or incombination. In the event that a domain contains a cysteine residueknown to form an inter-chain disulfide bridge to another PAPP-A subunitor to proMBP (see FIG. 8), it may be required that this cysteine ismutated to for example a serine or an alanine residue

Thus, possible boundary regions are between Cys-403 and Cys-499, betweenCys-828 and Cys-881, between Cys-1048 and Cys-1115, between Cys-1390 andCys-1396, between Cys-1459 and Cys-1464, between Cys-1521 and Cys-1525,between Cys-1590 and Cys-1595, between Cys-1646 and Cys-1653, andbetween Cys-1729 and Cys-1733 (numbering of preproPAPP-A2, as in FIGS. 1and 3).

Pharmaceutical Compositions

Identification of PAPP-A2 as the IGFBP-5 protease provides methods foraffecting growth and differentiation in vivo by using PAPP-A2 as atherapeutic target. Inhibitors of PAPP-A2 is believed to decrease theamount of bioavailable IGF-I and IGF-II. For example, inhibition ofPAPP-A2 activity can be useful in disorders such as restenosis,atherosclerosis, and fibrosis. Activators, or agents that increase theactivity of PAPP-A2, is believed to increase the amount of bioavailableIGF-I and IGF-II.

Agents that alter PAPP-A2 activity or that alter adherence of PAPP-A2 tocell surfaces can be incorporated into pharmaceutical compositions. Suchagents may be incorporated together with agents that alter PAPP-Aactivity or that alter adherence of PAPP-A to cell surfaces. Acombination of PAPP-A2 specific agents and PAPP-A specific agents may bemore effective than traditional agents directed against PAPP-A. There isalso provided a method of treatment comprising the step of administeringto an individual in need thereof a combination of PAPP-A2 specificagents and PAPP-A specific agents in pharmaceutically effective amounts.

As an example, an antibody such as anti-PAPP-A2 polyclonal ormonoclonal, can be formulated into a pharmaceutical composition byadmixture with pharmaceutically acceptable non-toxic excipients orcarriers. Such compounds and compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration maybe prepared as desired using standard methods.

Formulations for parenteral administration may contain as commonexcipients (i.e., pharmaceutically acceptable carriers) sterile water orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes, and the like. Inparticular, biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxethylene-polyoxypropylenecopolymers are examples of excipients for controlling the release of acompound of the invention in vivo. Other suitable parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain excipients such as lactose, ifdesired. Inhalation formulations may be aqueous solutions containing,for example, polyoxyethylene-9-lauryl ether, glycocholate anddeoxycholate, or they may be oily solutions for administration in theform of nasal drops. If desired, the compounds can be formulated as gelsto be applied intranasally. Formulations for parenteral administrationmay also include glycocholate for buccal administration

Medical Devices

The invention also features a medical device for placement in a patient(e.g., an implant) that includes an agent that inhibits or activatesPAPP-A2 protease activity. Suitable agents are readily identified usingthe methods described herein. The device can be impregnated with theagent or can be coated with the agent. Non-limiting examples ofinhibitors include an antibody such as anti-PAPP-A2 polyclonal ormonoclonal, or a metalloprotease inhibitor such as 1,10-phenanthroline.

IGFBP-5 protease activity of PAPP-A2 is potently inhibited by1,10-phenanthroline, but is not inhibited by tissue inhibitors of matrixmetalloproteases (TIMP'S). Other inhibitors include small molecules suchas derivatives of hydroxamic acid. Anti-PAPP-A2 polyclonal IgG may alsoinhibit IGF-dependent—or IGF-independent—IGFBP-5 specific PAPP-A2protease activity in HFCM in a dose-dependent manner.

In addition, polypeptides (i.e., any chain of amino acids, regardless oflength or post-translational modification), including modifiedpolypeptides, can function as inhibitors. Any inhibitor of the IGFBP-5protease activity of PAPP-A2 can be used for coating or impregnating amedical device according to the invention. Modified polypeptides includeamino acid substitutions, deletions, or insertions in the amino acidsequence as compared with a corresponding wild-type sequence, as well aschemical modifications. Although protease-resistant IGFBP-5 is not aninhibitor per se of the IGFBP-5 protease activity of PAPP-A2, similarresults are expected when it is used for coating or impregnating amedical device.

As an example, coating or impregnating the medical device with a PAPP-A2inhibitor, optionally in combination with a PAPP-A inhibitor, can helpprevent the development of restenosis following balloon angioplasty, orcan prevent a further increase in size of an atherosclerotic plaque.Coronary angioplasty with stent placement is currently the leadingtherapeutic approach for coronary atherosclerosis. An important goal ofangioplasty of coronary artery disease is to prevent both acute andchronic complications. Modern procedures are quite successful ineliminating immediate problems. Unfortunately, restenosis still occursin 20-30% of stented patients. No known pharmacological intervention isavailable to prevent the restenosis.

Without being bound by a particular mechanism, it is thought that anincrease in IGFBP-5 protease expression by coronary smooth muscle cellsprecedes neointimal formation in response to angioplasty in humans.

For example, enhanced PAPP-A2 activity can be useful for wound healing,fractures, osteoporosis, or ovulation. Osteoporosis or other conditionsof bone loss may benefit from increased bone formation and decreasedbone resorption. Agents that enhance PAPP-A2 activity can be, forexample, a modified IGF, i.e., an IGF analog.

Analogs include IGF polypeptides containing amino acid insertions,deletions or substitutions, as well as chemical modifications. Aminoacid substitutions can include conservative and non-conservative aminoacid substitutions. Conservative amino acid substitutions replace anamino acid with an amino acid of the same class, whereasnon-conservative amino acid substitutions replace an amino acid with anamino acid of a different class. Non-conservative substitutions resultin a change in the hydrophobicity of the polypeptide or in the bulk of aresidue side chain. In addition, non-conservative substitutions can makea substantial change in the charge of the polypeptide, such as reducingelectropositive charges or introducing electronegative charges. Examplesof non-conservative substitutions include a basic amino acid for anon-polar amino acid, or a polar amino acid for an acidic amino acid.Amino acid insertions, deletions and substitutions can be made usingrandom mutagenesis, site-directed mutagenesis, or other recombinanttechniques known in the art.

The medical device can be, for example, bone plates or bone screws thatare used to stabilize bones, or a stent, which typically is used withinthe body to restore or maintain the patency of a body lumen. Bloodvessels, for example, can become obstructed due to an atheroscleroticplaque that restricts the passage of blood. A stent typically has atubular structure defining an inner channel that accommodates flowwithin the body lumen. The outer walls of the stent engage the innerwalls of the body lumen. Positioning of a stent within an affected areacan help prevent further occlusion of the body lumen and permitcontinued flow. A stent typically is deployed by percutaneous insertionof a catheter or guide wire that carries the stent. The stent ordinarilyhas an expandable structure. Upon delivery to the desired site, thestent can be expanded with a balloon mounted on the catheter.Alternatively, the stent may have a biased or elastic structure that isheld within a sheath or other restraint in a compressed state. The stentexpands voluntarily when the restraint is removed. In either case, thewalls of the stent expand to engage the inner wall of the body lumen,and generally fix the stent in a desired position.

STATEMENTS OF INVENTION

In a first aspect the present invention relates to a purifiedpolynucleotide selected from the group consisting of

-   -   i) a polynucleotide comprising nucleotides 1 to 5376 of SEQ ID        NO:1, corresponding to the coding sequence of PAPP-A2, as        deposited with DSMZ under accession number DSM 13783; and    -   ii) a polynucleotide encoding a polypeptide having the amino        acid sequence as shown in SEQ ID NO:2; and    -   iii) a polynucleotide encoding a fragment of a polypeptide        encoded by polynucleotides (i) or (ii), wherein said fragment        -   a) has a proteolytic activity specific for Insulin Like            Growth Factor Binding Protein 5 (IGFBP-5), or a derivative            thereof, or any other substrate; and/or        -   b) is recognised by an antibody, or a binding fragment            thereof, which is capable of recognising a polypeptide            having the amino acid sequence as shown in SEQ ID NO:2;            and/or        -   c) competes with a polypeptide having the amino acid            sequence as shown in SEQ ID NO:2 for binding to a cell            surface receptor having an affinity for said polypeptide;            and    -   iv) a polynucleotide, the complementary strand of which        hybridizes, under stringent conditions, with a polynucleotide as        defined in any of (i), (ii) and (iii), said polynucleotide        encoding a polypeptide having the amino acid sequence as shown        in SEQ ID NO:2, or a fragment thereof, wherein said fragment        -   a) has a proteolytic activity specific at least for Insulin            Like Growth Factor Binding Protein 5 (IGFBP-5); and/or        -   b) is recognised by an antibody, or a binding fragment            thereof, which is capable of recognising a polypeptide            having the amino acid sequence as shown in SEQ ID NO:2;            and/or        -   c) competes with a polypeptide having the amino acid            sequence as shown in SEQ ID NO:2 for binding to a cell            surface receptor having an affinity for said polypeptide;            and    -   v) a polynucleotide comprising a nucleotide sequence which is        degenerate to the nucleotide sequence of a polynucleotide as        defined in any of (iii) and (iv),    -   and the complementary strand of such a polynucleotide.

A polynucleotide as used herein shall denote any naturally occurringpolynucleotide having any naturally occurring backbone structure, aswell as nucleotides known in the art as LNA (locked nucleic acid) andPNA (peptide nucleic acid).

In preferred embodiments the purified polynucleotide comprises thecoding sequence of PAPP-A2, nucleotides 1 to 5376, as shown in SEQ IDNO:1, or a nucleotide sequence encoding the amino acid sequence as shownin SEQ ID NO:2.

In another preferred embodiment the polynucleotide comprises anucleotide sequence encoding a fragment of the polypeptide having theamino acid sequence as shown in SEQ ID NO:2, wherein said fragment

-   -   a) has a proteolytic activity specific for Insulin Like Growth        Factor Binding Protein 5 (IGFBP-5), or a derivative thereof, or        any other substrate; and/or    -   b) is recognised by an antibody, or a binding fragment thereof,        which is capable of recognising a polypeptide having the amino        acid sequence as shown in SEQ ID NO:2; and/or    -   c) competes with a polypeptide having the amino acid sequence as        shown in SEQ ID NO:2 for binding to a cell surface receptor        having an affinity for said polypeptide

There is also provided a polynucleotide, the complementary strand ofwhich hybridizes, under stringent conditions, with a polynucleotideaccording to the invention.

Stringent conditions as used herein shall denote stringency as normallyapplied in connection with Southern blotting and hybridization asdescribed e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. Forsuch purposes it is routine practise to include steps ofprehybridization and hybridization. Such steps are normally performedusing solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50%formamide, 100 □g/ml denaturated salmon testis DNA (incubation for 18hrs at 42° C.), followed by washings with 2×SSC and 0.5% SDS (at roomtemperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS(incubation at 68° C. for 30 min), as described by Sambrook et al.,1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor),which is incorporated herein by reference.

The DNA sequences are used in a variety of ways. They may be used asprobes for identifying homologs of uHAse (e.g., homologs of huHAse).Mammalian homologs have substantial sequence similarity to one another,i.e. at least 75%, usually at least 90%, more usually at least 95%sequence identity. Sequence similarity is calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. 1990 J Mol Biol 215:403-10.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50.degree. C. and10.times.SSC (0.9 M saline/0.09 M sodium citrate) and remain bound whensubjected to washing at 55.degree. C. in 1.times.SSC. Sequence identitymay be determined by hybridization under high stringency conditions, forexample, at 50.degree. C. or higher and 0.1.times.SSC (9 mM saline/0.9mM sodium citrate). By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes. The source ofhomologous genes may be any species, e.g. Primate species, particularlyhuman; rodents, such as rats and mice, canines, felines, bovine, opines,equine, yeast, Drosophila, Caenhorabditis, etc.

In a further embodiment there is provided a polynucleotide comprising anucleotide sequence which is degenerate to a polynucleotide capable ofhybridising to SEQ ID NO:1, or a fragment thereof.

Degeneracy as used herein is defined in terms of the activity orfunctionality associated with the polypeptide expressed from saiddegenerate polynucleotide, said polynucleotide is either i) comprising aproteolytic activity specific at least for Insulin Like Growth FactorBinding Protein 5 (IGFBP-5); and/or ii) recognised by an antibody, or abinding fragment thereof, which is capable of recognising a polypeptidehaving the amino acid sequence as shown in SEQ ID NO:2; and/or iii)competing with a polypeptide having the amino acid sequence as shown inSEQ ID NO:2 for binding to a cell surface receptor having an affinityfor said polypeptide.

In a further embodiment there is provided a polynucleotide comprisingthe complementary strand of a polynucleotide according to the invention.

The polynucleotide according to the invention may be operably linked toa further polynucleotide comprising nucleic acid residues correspondingto the 3′ untranslated region of PAPP-A2, or a fragment thereof. As usedherein the 3′ untranslated region comprises nucleic acid residues 5377to 8527 of SEQ ID NO:1.

There is also provided a recombinant DNA molecule in the form of anexpression vector comprising an expression signal operably linked to apolynucleotide according to the invention.

In a further embodiment there is provided a host organism transfected ortransformed with the polynucleotide according to the invention, or thevector according to the invention. The host organism is preferably amammalian organism such as e.g. a mammalian cell line. However, amicrobial eukaryote such as yeast or fungi may also be used, as may amicrobial prokaryote such as Bacillus or E. coli. The person skilled inthe art will know how to select expression signals, including leadersequences and/or signal peptides suitable for expression in a givencell. The person skilled in the art will also know how to determine thelevel of expression in a given cell by using standard molecular biologytechniques.

In a further aspect the invention relates to an isolated polypeptidecomprising or essentially consisting of the amino acid sequence of SEQID NO:2, or a fragment thereof, wherein said fragment

-   -   a) has a proteolytic activity specific at least for Insulin Like        Growth Factor Binding Protein 5 (IGFBP-5); and/or    -   b) is recognised by an antibody, or a binding fragment thereof,        which is capable of recognising a polypeptide having the amino        acid sequence as shown in SEQ ID NO:2; and/or    -   c) competes with a polypeptide having the amino acid sequence as        shown in SEQ ID NO:2 for binding to a cell surface receptor with        an affinity for said polypeptide.

In one preferred embodiment of the invention there is also providedvariants of SEQ ID NO:2, and variants of fragments thereof. Variants aredetermined on the basis of their degree of identity or their homologywith a predetermined amino acid sequence, said predetermined amino acidsequence being SEQ ID NO:2, or, when the variant is a fragment, afragment of SEQ ID NO:2.

Accordingly, variants preferably have at least 75% sequence identity,for example at least 80% sequence identity, such as at least 85%sequence identity, for example at least 90% sequence identity, such asat least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with thepredetermined sequence.

Variants are also determined based on a predetermined number ofconservative amino acid substitutions as defined herein below.Conservative amino acid substitution as used herein relates to thesubstitution of one amino acid (within a predetermined group of aminoacids) for another amino acid (within the same group), wherein the aminoacids exhibit similar or substantially similar characteristics.

Within the meaning of the term “conservative amino acid substitution” asapplied herein, one amino acid may be substituted for another within thegroups of amino acids indicated herein below:

-   -   i) Amino acids having polar side chains (Asp, Glu, Lys, Arg,        His, Asn, Gln, Ser, Thr, Tyr, and Cys)    -   ii) Amino acids having non-polar side chains (Gly, Ala, Val,        Leu, Ile, Phe, Trp, Pro, and Met)    -   iii) Amino acids having aliphatic side chains (Gly, Ala Val,        Leu, Ile)    -   iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His,        Pro)    -   v) Amino acids having aromatic side chains (Phe, Tyr, Trp)    -   vi) Amino acids having acidic side chains (Asp, Glu)    -   vii) Amino acids having basic side chains (Lys, Arg, His)    -   viii) Amino acids having amide side chains (Asn, Gln)    -   ix) Amino acids having hydroxy side chains (Ser, Thr)    -   x) Amino acids having sulphor-containing side chains (Cys, Met),    -   xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,        Thr)    -   xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and    -   xiii) Hydrophobic amino acids (Leu, Ile, Val)

Accordingly, a variant or a fragment thereof according to the inventionmay comprise, within the same variant of the sequence or fragmentsthereof, or among different variants of the sequence or fragmentsthereof, at least one substitution, such as a plurality of substitutionsintroduced independently of one another.

It is clear from the above outline that the same variant or fragmentthereof may comprise more than one conservative amino acid substitutionfrom more than one group of conservative amino acids as defined hereinabove.

The addition or deletion of an amino acid may be an addition or deletionof from 2 to 10 amino acids, such as from 10 to 20 amino acids, forexample from 20 to 30 amino acids, such as from 40 to 50 amino acids.However, additions or deletions of more than 50 amino acids, such asadditions from 10 to 100 amino acids, addition of 100 to 150 aminoacids, addition of 150-250 amino acids, are also comprised within thepresent invention.

The polypeptide fragments according to the present invention, includingany functional equivalents thereof, may in one embodiment comprise lessthan 250 amino acid residues, such as less than 240 amino acid residues,for example less than 225 amino acid residues, such as less than 200amino acid residues, for example less than 180 amino acid residues, suchas less than 160 amino acid residues, for example less than 150 aminoacid residues, such as less than 140 amino acid residues, for exampleless than 130 amino acid residues, such as less than 120 amino acidresidues, for example less than 110 amino acid residues, such as lessthan 100 amino acid residues, for example less than 90 amino acidresidues, such as less than 85 amino acid residues, for example lessthan 80 amino acid residues, such as less than 75 amino acid residues,for example less than 70 amino acid residues, such as less than 65 aminoacid residues, for example less than 60 amino acid residues, such asless than 55 amino acid residues, for example less than 50 amino acidresidues.

“Functional equivalency” as used in the present invention is accordingto one preferred embodiment established by means of reference to thecorresponding functionality of a predetermined fragment of the sequence.More specifically, functional equivalency is to be understood as theability of a polypeptide fragment to exert IGFBP-5 specific proteaseactivity and/or to be recognised by an antibody capable of recognisingPAPP-A2 and/or to compete with PAPP-A2 for binding to a receptor havingaffinity for PAPP-A2.

Functional equivalents or variants of PAPP-A2 will be understood toexhibit amino acid sequences gradually differing from the preferredpredetermined PAPP-A2 sequence, as the number and scope of insertions,deletions and substitutions including conservative substitutionsincreases. This difference is measured as a reduction in homologybetween the preferred predetermined sequence and the fragment orfunctional equivalent.

All fragments or functional equivalents of SEQ ID NO:2 are includedwithin the scope of this invention, regardless of the degree of homologythat they show to a preferred predetermined sequence of PAPP-A2 asreported herein. The reason for this is that some regions of PAPP-A2 aremost likely readily mutatable, or capable of being completely deleted,without any significant effect on the binding activity of the resultingfragment.

A functional variant obtained by substitution may well exhibit some formor degree of native PAPP-A2 activity, and yet be less homologous, ifresidues containing functionally similar amino acid side chains aresubstituted. Functionally similar in this respect refers to dominantcharacteristics of the side chains such as hydrophobic, basic, neutralor acidic, or the presence or absence of steric bulk. Accordingly, inone embodiment of the invention, the degree of identity is not aprincipal measure of a fragment being a variant or functional equivalentof a preferred predetermined fragment according to the presentinvention.

The homology between amino acid sequences may be calculated using wellknown algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50,BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM80, BLOSUM 85, or BLOSUM 90.

Fragments sharing at least some homology with fragments of SEQ ID NO:2are to be considered as falling within the scope of the presentinvention when they are at least about 90 percent homologous, forexample at least 92 percent homologous, such as at least 94 percenthomologous, for example at least 95 percent homologous, such as at least96 percent homologous, for example at least 97 percent homologous, suchas at least 98 percent homologous, for example at least 99 percenthomologous with said fragments of SEQ ID NO:2. According to oneembodiment of the invention the homology percentages refer to identitypercentages.

Additional factors that may be taken into consideration when determiningfunctional equivalence according to the meaning used herein are i) theability of antisera to detect a PAPP-A2 fragment according to thepresent invention, or ii) the ability of the functionally equivalentPAPP-A2 fragment to compete with PAPP-A2 in a binding assay. One methodof determining a sequence of immunogenically active amino acids within aknown amino acid sequence has been described by Geysen in U.S. Pat. No.5,595,915 and is incorporated herein by reference.

A further suitably adaptable method for determining structure andfunction relationships of peptide fragments is described by U.S. Pat.No. 6,013,478, which is herein incorporated by reference. Also, methodsof assaying the binding of an amino acid sequence to a receptor moietyare known to the skilled artisan.

Conservative substitutions may be introduced in any position of apreferred predetermined fragment of SEQ ID NO:2, and it may also bedesirable to introduce non-conservative substitutions in any one or morepositions.

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of PAPP-A2 would for example i) differsubstantially in polarity, for example a residue with a non-polar sidechain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) substituted for aresidue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, orGln or a charged amino acid such as Asp, Glu, Arg, or Lys, orsubstituting a charged or a polar residue for a non-polar one; and/orii) differ substantially in its effect on polypeptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Variants obtained by substitution of amino acids may in one preferredembodiment be made based upon the hydrophobicity and hydrophilicityvalues and the relative similarity of the amino acid side-chainsubstituents, including charge, size, and the like. Exemplary amino acidsubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

In addition to the variants described herein, sterically similarvariants may be formulated to mimic the key portions of the variantstructure and that such compounds may also be used in the same manner asthe variants of the invention. This may be achieved by techniques ofmodelling and chemical designing known to those of skill in the art. Itwill be understood that all such sterically similar constructs fallwithin the scope of the present invention.

In a further embodiment the present invention relates to functionalcomprising substituted amino acids having hydrophilic or hydropathicindices that are within +/−2.5, for example within +/−2.3, such aswithin +/−2.1, for example within +/−2.0, such as within +/−1.8, forexample within +/−1.6, such as within +/−1.5, for example within +/−1.4,such as within +/−1.3 for example within +/−1.2, such as within +/−1.1,for example within +/−1.0, such as within +/−0.9, for example within+/−0.8, such as within +/−0.7, for example within +/−0.6, such as within+/−0.5, for example within +/−0.4, such as within +/−0.3, for examplewithin +/−0.25, such as within +/−0.2 of the value of the amino acid ithas substituted.

The importance of the hydrophilic and hydropathic amino acid indices inconferring interactive biologic function on a protein is well understoodin the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101,each incorporated herein by reference).

The amino acid hydropathic index values as used herein are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5) (Kyte & Doolittle, 1982).

The amino acid hydrophilicity values are: arginine (+3.0); lysine(+3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4) (U.S. Pat. No.4,554,101).

In addition to the peptidyl compounds described herein, stericallysimilar compounds may be formulated to mimic the key portions of thepeptide structure and that such compounds may also be used in the samemanner as the peptides of the invention. This may be achieved bytechniques of modelling and chemical designing known to those of skillin the art. For example, esterification and other alkylations may beemployed to modify the amino terminus of, e.g., a di-arginine peptidebackbone, to mimic a tetra peptide structure. It will be understood thatall such sterically similar constructs fall within the scope of thepresent invention.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed within the present invention. Functional equivalentsalso comprise glycosylated and covalent or aggregative conjugates formedwith the same or other PAPP-A2 fragments and/or PAPP-A2 molecules,including dimers or unrelated chemical moieties. Such functionalequivalents are prepared by linkage of functionalities to groups whichare found in fragment including at any one or both of the N- andC-termini, by means known in the art.

Functional equivalents may thus comprise fragments conjugated toaliphatic or acyl esters or amides of the carboxyl terminus, alkylaminesor residues containing carboxyl side chains, e.g., conjugates toalkylamines at aspartic acid residues; O-acyl derivatives of hydroxylgroup-containing residues and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g. conjugates withfMet-Leu-Phe or immunogenic proteins. Derivatives of the acyl groups areselected from the group of alkyl-moieties (including C3 to C10 normalalkyl), thereby forming alkanoyl species, and carbocyclic orheterocyclic compounds, thereby forming aroyl species. The reactivegroups preferably are bifunctional compounds known per se for use incross-linking proteins to insoluble matrices through reactive sidegroups.

Covalent or aggregative functional equivalents and derivatives thereofare useful as reagents in immunoassays or for affinity purificationprocedures. For example, a fragment of PAPP-A2 according to the presentinvention may be insolubilized by covalent bonding to cyanogenbromide-activated Sepharose by methods known per se or adsorbed topolyolefin surfaces, either with or without glutaraldehydecross-linking, for use in an assay or purification of anti-PAPP-A2antibodies or cell surface receptors. Fragments may also be labelledwith a detectable group, e.g., radioiodinated by the chloramine Tprocedure, covalently bound to rare earth chelates or conjugated toanother fluorescent moiety for use in e.g. diagnostic assays.

Mutagenesis of a preferred predetermined fragment of PAPP-A2 can beconducted by making amino acid insertions, usually on the order of aboutfrom 1 to 10 amino acid residues, preferably from about 1 to 5 aminoacid residues, or deletions of from about from 1 to 10 residues, such asfrom about 2 to 5 residues.

In one embodiment the fragment of PAPP-A2 is synthesised by automatedsynthesis. Any of the commercially available solid-phase techniques maybe employed, such as the Merrifield solid phase synthesis method, inwhich amino acids are sequentially added to a growing amino acid chain.(See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963).

Equipment for automated synthesis of polypeptides is commerciallyavailable from suppliers such as Applied Biosystems, Inc. of FosterCity, Calif., and may generally be operated according to themanufacturer's instructions. Solid phase synthesis will enable theincorporation of desirable amino acid substitutions into any fragment ofPAPP-A2 according to the present invention. It will be understood thatsubstitutions, deletions, insertions or any subcombination thereof maybe combined to arrive at a final sequence of a functional equivalent.Insertions shall be understood to include amino-terminal and/orcarboxyl-terminal fusions, e.g. with a hydrophobic or immunogenicprotein or a carrier such as any polypeptide or scaffold structurecapable as serving as a carrier.

Oligomers including dimers including homodimers and heterodimers offragments of PAPP-A2 according to the invention are also provided andfall under the scope of the invention. PAPP-A2 functional equivalentsand variants can be produced as homodimers or heterodimers with otheramino acid sequences or with native PAPP-A2 sequences. Heterodimersinclude dimers containing immunoreactive PAPP-A2 fragments as well asPAPP-A2 fragments that need not have or exert any biological activity.

PAPP-A2 fragments according to the invention may be synthesised both invitro and in vivo. Method for in vitro synthesis are well known, andmethods being suitable or suitably adaptable to the synthesis in vivo ofPAPP-A2 are also described in the prior art. When synthesized in vivo, ahost cell is transformed with vectors containing DNA encoding PAPP-A2 ora fragment thereof. A vector is defined as a replicable nucleic acidconstruct. Vectors are used to mediate expression of PAPP-A2. Anexpression vector is a replicable DNA construct in which a nucleic acidsequence encoding the predetermined PAPP-A2 fragment, or any functionalequivalent thereof that can be expressed in vivo, is operably linked tosuitable control sequences capable of effecting the expression of thefragment or equivalent in a suitable host. Such control sequences arewell known in the art.

Cultures of cells derived from multicellular organisms representpreferred host cells. In principle, any higher eukaryotic cell cultureis workable, whether from vertebrate or invertebrate culture. Examplesof useful host cell lines are VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.Preferred host cells are eukaryotic cells known to synthesize endogenousPAPP-A2. Cultures of such host cells may be isolated and used as asource of the fragment, or used in therapeutic methods of treatment,including therapeutic methods aimed at promoting or inhibiting a growthstate, or diagnostic methods carried out on the human or animal body.

In particular embodiments the present invention relates to a polypeptidefragment according to the invention, wherein the PAPP-A2 fragmentcomprises or essentially consists of amino acid residues 234 to 1791corresponding to the mature part of PAPP-A2, including any processingvariants thereof.

Processing variants are variants resulting from alternative processingevents, possibly processing events catalysed by any protease including,but not limited to, a signal peptidase and a furin. One putativecleavage site is located after position 233 is described herein below indetail. Another putative cleavage site is located after the motif RQRR(position 196-199 in the amino acid sequence of PAPP-A2). Processingvariants shall be understood to comprise variants arising fromprocessing in vivo when PAPP-A2 is expressed in human or animal tissue,sera or body fluids.

Mature PAPP-A2 amino acids sequences essentially consisting of themature sequence designated in SEQ ID NO:2 (amino acid residues 234 to1791) shall be understood in one embodiment to comprise this part of thesequence lacking between 1 to about 10 N-terminal amino acids orC-terminal amino acids, preferably 1 to 10 N-terminal amino acids, suchas 2 to 8 N-terminal acids, for example 3 to 6 N-terminal amino acids.

Also included in the definition of essentially consisting of as usedherein shall be the mature sequence designated in SEQ ID NO:2 (aminoacid residues 234 to 1791) having in addition thereto an additional 1 toabout 10 N-terminal amino acids or C-terminal amino acids, preferably 1to 10 N-terminal amino acids, such as 2 to 8 N-terminal acids, forexample 3 to 6 N-terminal amino acids. This definition of essentiallyconsisting of shall also apply in other aspects and is not restricted tobeing used in connection with a particular part of PAPP-A2. Thedefinition shall also apply to other processes PAPP-A2 polypeptidesincluding polypeptides arising from alternative processing in tissue,sera or body fluids other than the ones from where the processed PAPP-A2has originally been isolated.

Additionally preferred fragments comprise or essentially consists ofamino acid residues 1 to 233 corresponding to the prepro part ofPAPP-A2, of amino acid residues 23 to 233 corresponding to the pro partof PAPP-A2, of amino acid residues 1 to 22 corresponding to the signalpeptide or leader sequence of PAPP-A2, and to such sequences operablylinked to the mature part of PAPP-A2 corresponding to amino acidresidues 234 to 1791 of SEQ ID NO:2.

There is also provided recombinant PAPP-A2 polypeptide, or a fragmentthereof, wherein preferably the polypeptide is free of human proteins,or other proteins natively associated with said polypeptide.

In a further aspect there is provided a composition comprising i) apolynucleotide according to the invention, and/or ii) a vector accordingto the invention, and/or iii) a host organism according to theinvention, and/or iv) a polypeptide according to the invention, incombination with a physiologically acceptable carrier.

In yet another aspect there is provided a pharmaceutical compositioncomprising i) a polynucleotide according to the invention, and/or ii) avector according to the invention, and/or iii) a host organism accordingto the invention, and/or iv) a polypeptide according to the invention,in combination with a pharmaceutically acceptable carrier.

The invention further pertains to a method for producing an antibodywith specificity for a PAPP-A2 polypeptide according to the invention,or a fragment thereof, said method comprising the steps of

-   -   i) providing a host organism,    -   ii) immunizing the host organism with the polypeptide according        to claim 10, and    -   iii) obtaining said antibody.

There is also provided monoclonal antibodies and polyclonal antibodieshaving specific binding affinity for a PAPP-A2 polypeptide according tothe invention, or a fragment thereof. The antibody is preferably amonoclonal.

In a further aspect there is provided a method for producing a PAPP-A2polypeptide according to the invention, said method comprising the stepsof

-   -   i) providing a suitable host organism, preferably a mammalian        cell,    -   ii) transfecting or transforming the host organism provided in        step i) with a polynucleotide according to the invention, or a        vector according to the invention,    -   iii) culturing the host organism obtained in step ii) under        conditions suitable for expression of the polypeptide encoded by        the polynucleotide or the vector; and optionally    -   iv) isolating from the host organism the polypeptide resulting        from recombinant expression by the host organism.

In a still further aspect of the invention there is provided a methodfor inhibiting and/or reducing the expression of PAPP-A2 in a cell bymeans of anti-sense technology, said method comprising the steps of

-   -   i) providing an anti-sense polynucleotide according to the        invention,    -   ii) transfecting or transforming a cell capable of expressing        PAPP-A2 with said anti-sense polynucleotide provided in step i),    -   iii) culturing the cell obtained in step ii) under conditions        suitable for hybridization of the polynucleotide provided in        step i) to a complementary polynucleotide in said cell involved        in the expression of PAPP-A2, and    -   iv) inhibiting and/or reducing the expression of PAPP-A2 in said        cell.

The antisense polynucleotide and the complementary polynucleotide may beco-expressed from distinct polynucleotide molecules or they may beexpressed from the same molecule. As an alternative to hybridization,the method may include the use of reverse transcriptase PCR technology(rt PCT technology).

In yet another aspect of the invention there is provided a method fordetecting PAPP-A2, or measuring the level of PAPP-A2, in a biologicalsample obtained from an individual, said method comprising the steps of

-   -   i) obtaining a biological sample from said individual,    -   ii) detecting PAPP-A2 in said sample by detecting        -   a) a PAPP-A2 polypeptide, or a fragment thereof, and/or        -   b) a polynucleotide in the form of mRNA originating from            PAPP-A2 expression, and/or        -   c) PAPP-A2 specific protease activity, preferably IGFBP-5            protease activity, or proteolytic activity directed against            a derivative of IGFBP-5.

The method may comprise the further step of comparing the PAPP-A2 or thelevel of PAPP-A2 detected in step ii) with a predetermined valueselected from the group consisting of

-   -   a) a predetermined amount and/or concentration of PAPP-A2;        and/or    -   b) a predetermined amount and/or concentration of PAPP-A2 mRNA;        and/or    -   c) a predetermined PAPP-A2 specific protease activity.

The predetermined value in one embodiment will be indicative of a normalphysiological condition of said individual.

The biological sample is preferably selected from the group consistingof blood, urine, pleural fluid, oral washings, tissue biopsies, andfollicular fluid.

When the level of PAPP-A2 is measured as an amount of PAPP-A2 protein,the PAPP-A2 protein is preferably measured by immunochemical analysiswherein PAPP-A2 protein is detected by at least one monoclonal antibody.PAPP-A2 protein may also be detected in a complex comprising at leastone additional component, preferably a polypeptide such as, but notlimited to, pro-MBP (pro-Major-Basic Protein). PAPP-A2 may also bedetected as a PAPP-A2 monomer or as a PAPP-A2 dimer.

Further aspects of the invention relates to a method of diagnosing aclinical condition in an individual, said method comprising the steps of

-   -   i) performing a method for detecting PAPP-A2 or measuring the        level of PAPP-A2, and    -   ii) diagnosing the clinical condition.

The clinical condition is preferably a fetal abnormality such as, butnot limited to, a fetal abnormality selected from the group consistingof Trisomy 21, Trisomy 18, Trisomy 13, and Open Spina Bifida.

Additional fetal abnormalities capable of being diagnosed according tothe invention is ectopic pregnancy, open spina bifida, neural tubedefects, ventral wall defects, Edwards Syndrome, Pateaus Syndrome,Turner Syndrome, Monosomy X or Kleinfelter's Syndrome.

In another aspect the clinical condition is an altered growth stateselected from the group consisting of a growth promoting state and agrowth inhibiting state, including, but not limited to, restenosis,atherosclerosis, wound healing, fibrosis, myocardial infarction,osteoporosis, rheumatoid arthritis, multiple myeloma, or cancer.

In a yet further aspect of the invention there is provided a method fordetecting expression of a polynucleotide according to the invention in abiological sample, said method comprising the steps of

-   -   i) providing a biological sample putatively containing a        polynucleotide according to the invention, and    -   ii) contacting the biological sample with a polynucleotide        comprising a strand that is i) complementary to the        polynucleotide according to the invention and ii) capable of        hybridizing thereto, and    -   iii) allowing hybridization to occur, and    -   iv) detecting the hybridization complex obtained in step iii),    -   wherein the presence of the hybridization complex is indicative        of the expression in the biological sample of the polynucleotide        according to the invention, or a fragment thereof.

In a still further aspect of the invention there is provided a methodfor identifying an agent inhibiting the protease activity of PAPP-A2,said method comprising the steps of

-   -   i) incubating a) the polypeptide according to the invention, or        a fragment thereof, and b) a predetermined substrate for said        polypeptide or fragment, and c) a putative inhibitory agent, and    -   ii) determining if proteolysis of said substrate is inhibited.

The substrate preferably comprises a polypeptide that may be aninternally quenched fluorescent peptide. One preferred substratecomprises or essentially consists of IGFBP-5, or a fragment thereof.

The invention also pertains to an inhibitory agent obtainable accordingto such a method for identifying an agent inhibiting the proteaseactivity of PAPP-A2.

There is also provided the use of such provided inhibitory agents in themanufacture of a medicament for treating a clinical condition in anindividual in need of such treatment.

In a still further aspect the invention pertains to a method foridentifying an agent capable of enhancing the protease activity ofPAPP-A2, said method comprising the steps of

-   -   i) incubating a) the polypeptide according to the invention, or        a fragment thereof, and b) a predetermined substrate for said        polypeptide, and c) a putative enhancer agent, and    -   ii) determining if proteolysis of said substrate is enhanced.

The substrate preferably comprises a polypeptide including an internallyquenched fluorescent peptide. IGFBP-5, or a fragment thereof, isparticularly preferred as a substrate.

There is also provided an enhancing agent obtainable according to themethod for identifying an agent capable of enhancing the proteaseactivity of PAPP-A2, and the invention also pertains to the use of suchenhancing agents in the manufacture of a medicament for treating aclinical condition in an individual in need of such treatment.

In yet another aspect there is provided a method of treatment by therapyof an individual, said method comprising the step of administrating tosaid individual i) a pharmaceutical composition according to theinvention, and/or ii) the inhibitory agent according to the invention,and/or the enhancing agent according to the invention.

In a still further aspect there is provided a method for purification ofPAPP-A2 or complexes of PAPP-A2 with other proteins, said methodcomprising the steps of

-   -   i) providing a polyclonal or monoclonal antibody with specific        binding affinity for a polypeptide according to the invention,        or a fragment thereof, and    -   ii) purifying PAPP-A2, or a fragment thereof, by means of        affinity chromatography.

It is understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

EXAMPLES Example 1 Identification of a Nucleotide Sequence EncodingPAPP-A2

Accession numbers (ANs) given in this text refer to sequences depositedin GenBank or other biological sequence databases. ANs are usedinterchangeable with the protein or nucleotide sequences deposited underthe given AN.

Searching public nucleotide databases for DNA sequences with homology toPAPP-A ((Kristensen et al., 1994, Biochemistry 33, 1592-8), AN CAA48341)when translated into polypeptide sequence revealed two genomic cloneswith the ANs AL031734 and AL031290. Both originate from the humanchromosome 1 (1q24). The search was performed against the “nr”collection of databases using the program tblastn atncbi.nlm.nih.gov/BLAST/ with default settings. In this example, PAPP-Ais numbered with the N-terminal Glu as residue 1, as in (Kristensen etal., 1994, Biochemistry 33, 1592-8). In the deposited sequence record(AN X68280) this Glu is residue 5.

The sequence reported in AL031734 contains 168835 base pairs. Twononcontiguous sequence stretches (nt. 103432-103566, and 140846-141919)of the total sequence together aligned with residues 16-59, and 59-413of the PAPP-A polypeptide sequence when translated. The sequencereported in AL031290 contains 121780 base pairs. Four noncontiguoussequence stretches (nt. 10209-10358, 11752-11901, 20531-20463, and60536-60652) of the total sequence together aligned with residues1313-1362, 1376-1425, 1457-1479, and 1470-1506 of the PAPP-A polypeptidesequence when translated. The sequence stretches between the codingregions of both of the genomic sequences represent noncoding genomic DNA(introns) or coding regions that do not align.

Based on these findings, we hypothesized the existence of a novelprotein, PAPP-A2, with homology to PAPP-A. It was then established thecomplete coding sequence of the regions of PAPP-A2 that were partiallycovered by the two genomic sequences reported in AL031734 and AL031290.We denote those contiguous sequences hom-N and hom-C, respectively (FIG.2). But first, we established the existence of a coding cDNA sequencethat also showed homology to PAPP-A, and that connected the sequence ofhom-N and hom-C (FIG. 2). All essential primers used are described inTable 1. The entire cDNA sequence encoding the 1791-residuepreproPAPP-A2 is shown in FIG. 1. Standard cloning techniques were used,and all DNA constructs were analyzed by sequencing. The methodology usedis described below. The name PAPP-A2 is used for the protein encoded bythis DNA sequence.

Cloning of a contiguous coding cDNA stretch corresponding to themidregion between hom-N and hom-C: To obtain the midregion (FIG. 2),cDNA was synthesized using human placental mRNA as a template and aprimer, RT-N-mid, derived from AL031290 (Table 1, FIG. 2). This cDNA wasused as a template in a PCR to obtain a cDNA corresponding to themidregion of the hypothesized PAPP-A2. PCR primers were PR-mid5 andPR-mid3 (Table 1, FIG. 2). The coding sequence of the midregion obtainedcorresponds to residues 665-1572 of FIG. 3 (SEQ ID NO:1), a total of 908amino acids.

TABLE 1 Locations of primers used for reverse transcription or PCR.The primers are listed in the order of their use. NAME SOURCE^(a)Nt. NUMBERS^(b) SEQUENCE^(c) RT-N-mid: AL031290  10262-10281,GCTCACACACCACAGGAATG*  (4770-4789) (SEQ ID NO: 4) PR-mid5: AL031734141874-141894, GGCTGATGTGCGCAAGACCTG  (1947-1967) (SEQ ID NO: 5)PR-mid3: AL031290  10208-10229, GCATTGTATCTTCAGGAGCTTG*  (4716-4737)(SEQ ID NO: 6) PR-N5: AL031734 102606-102628, GAAGTTGACTTCTGGTTCTGTAG(—) (SEQ ID NO: 7) PR-N3: — —, CCCTGGGAAGCGAGTGAAGCC*  (2380-2400)(SEQ ID NO: 8) RT-C: AL031290  62982-63006, GCATTTCTTATAAGATCCTTCATGC*(—) (SEQ ID NO: 9) PR-C5: — —, GACAGCTGTCCGTCATTGCTGC  (4180-4201)(SEQ ID NO: 10) PR-C3: AL031290  62876-62897, CTTACTGCCTCTGAGGCAGTGG*(—) (SEQ ID NO: 11) ^(a)Accession numbers of the relevant genomic clonesare given. Primers PR-N3 and PR-C5 were located in the sequenceconnecting hom-N and hom-C, and are therefore not represented in thedatabases. ^(b)Nucleotide numbers refer to the numbering of thesequences as reported in the file with the relevant accession number. Inparentheses are given the corresponding numbers of SEQ ID NO: 1 (FIG.1), except for primers PR-N5, RT-C and PR-C3, not within this sequence.^(c)Sequences are actual primer sequences (orientation 5′-to-3′).Sequences marked with an asterisk are complementary to the databasesequences or the sequence given in FIG. 1.

Cloning of a contiguous coding cDNA stretch corresponding to theN-terminal end of PAPP-A2 (hom-N): Manual inspection of the genomicsequence AL031734 revealed that the open reading frame of the sequencestretch corresponding to PAPP-A residues 16-59 continued further in the5′ direction: Nt. 102646-103566 encodes a polypeptide sequence of 307residues that starts with a methionine residue. Based on this finding,the cDNA used to obtain the midregion (placental mRNA primed withRT-N-mid, as detailed above) was used as a template in a PCR to obtainthe contiguous cDNA of hom-N. PCR primers were: PR-N5 and PR-N3 (Table1, FIG. 2).

Cloning of a contiguous coding cDNA stretch corresponding to theC-terminal end of PAPP-A2 (hom-C): Searching available databases (usingthe program blastn at ncbi.nlm.nih.gov/BLAST/with default settings) forhuman EST sequences matching the genomic sequence of AL031290 revealedan EST sequence overlapping with some of the coding regions of AL031290already defined by the stretch nt. 60536-60652 (cf. above). Nt.62790-62995 of AL031290 also matched the sequence of the human ESTsequence AA368081 originating from placenta.

When translated into polypeptide sequence, this EST sequence showedhomology to the C-terminal end of PAPP-A. Further, a stop codon waspresent within the coding sequence corresponding to amino acid 1537 ofPAPP-A. That is, PAPP-A2 does not extend C-terminally beyond PAPP-A whenthe two sequences are aligned. Based on this, cDNA was synthesized usinghuman placental mRNA as a template and a primer originating fromAL031290 (Table 1). This cDNA was used as a template in a PCR to obtainthe contiguous cDNA of hom-C using PCR primers PR-C5 and PR-C3 (Table 1,FIG. 2).

All PCRs were carried out with Pfu polymerase (Stratagene). The threeoverlapping PAPP-A2 cDNA fragments (hom-N, the novel midregion, andhom-C) were all cloned into the vector pCR-BluntII-TOPO (Invitrogen).Several clones were sequenced in both orientations. The constructs arereferred to as p2N, p2Mid, and p2C, respectively. The entire nucleotidesequence encoding PAPP-A2 is shown in FIG. 1 (and SEQ ID NO:1).

Example 2 Analyses of the Nucleotide and Amino Acid Sequence of PAPP-A2

Of the 1547 residues of mature PAPP-A, 708 residues (45.8%) areidentical in preproPAPP-A2. There is no significant degree of identitybetween the prepro portion of PAPP-A and the remaining (N-terminal)portion of PAPP-A2 (FIG. 3). In this example, PAPP-A is numberedaccording to ((Haaning et al., 1996, Eur J Biochem 237, 159-63),AAC50543).

The sequence motifs recognized in PAPP-A (Kristensen et al., 1994,Biochemistry 33, 1592-8) are also present PAPP-A2: An elongated zincbinding consensus sequence, three lin-notch repeats (LNR1-3), and fiveshort consensus repeats (SCR1-5) (FIG. 3). Further, all 82 cysteineresidues of PAPP-A are conserved between the two proteins, and anadditional 4 cysteines are present in the PAPP-A2 polypeptide sequence.

Example 3 Identification of Human EST Sequences Originating from thePAPP-A2 mRNA

A cluster of EST sequences matching the genomic sequence of AL031290were identified around nt 64000-66000 of AL031290, startingapproximately 1.2 kb from the end of the PAPP-A2 encoding sequence. Theexistence of mRNA connecting the coding region of PAPP-A2 and thiscluster was verified in a PCR using primers from AL031290(5′-GGAAAGAGCAGAGTTCACCCAT-3′ (SEQ ID NO:12), nt. 64900-64879 ofAL031290) and the PAPP-A2 encoding sequence (5′-CCGTCTTAGTCCACTGCATCC-3′(SEQ ID NO:13), nt. 20499-20519 of AL031290, nt 5171-5191 of AF311940),and oligo-dT primed placental cDNA as a template (Overgaard et al.,1999, Biol Reprod 61, 1083-9). As expected, the size of the resultingproduct was 2.2 kb, further demonstrating the existence of a PAPP-A2mRNA with a 3′UTR of about 3 kb. The distribution among tissues is shownin Table 2.

TABLE 2 Expression of PAPP-A2 mRNA in human tissues evaluated byavailable EST sequences^(a). Tissue of origin Number of ESTs found Humanplacenta 38 Pregnant uterus 21 Fetal liver/spleen 11 Kidney 5Retina/Fetal retina 3 Corneal stroma 2 Fetal heart 2 Gessler Wilms tumor2 Other tissues^(b) 14 ^(a)Using the blast algorithm (Altschul et al.,1997, Nucleic Acids Res 25, 3389-402), a total of 98 human EST sequenceswere identified that matched the 3′UTR of the PAPP-A2 mRNA sequence. Thedistribution among tissues is based on the annotations of individualdatabase entries (not listed). ^(b)EST sequences originated from poolsof tissue, or from tissue represented by only one EST sequence.

Example 4 Expression in Mammalian Cells of Recombinant PAPP-A2 andVariants of PAPP-A2

The following plasmid constructs were made:

a) pPA2: The cDNA sequence of pre-pro-PAPP-A2 encoding amino acids1-1791 in expression vector pcDNA3.1+.

b) pPA2-KO: As pPA2, but Glu-734 of the active site of PAPP-A2substituted with a Gln residue (E734Q).

c) pPA2-mH: The expression vector pcDNA3.1/Myc-His(−)A containing thecDNA sequence of pre-pro-PAPP-A2 encoding amino acids 1-1791, notfollowed by a stop codon, but rather a c-myc and a His tag.

d) pPA2-KO-mH: As pPA2-mH, but with the E734Q substitution of pPA2-KO.

The three overlapping PAPP-A2 cDNA fragments (hom-N, the midregion, andhom-C) were used for the construction of a single contiguous cDNAsequence encoding PAPP-A2. The overlapping fragments were all containedin the vector pCR-BluntII-TOPO (Invitrogen) and referred to as p2N,p2Mid, and p2C, as detailed above (example 6.1). Clones of p2N and p2Cwere selected that had the proper orientation of the cDNA insert.

Construction of pPA2: The NotI-BamHI fragment was excised from p2C andcloned into pBluescriptIISK+ (Stratagene) to obtain p2CBlue. TheNotI-SpeI fragment was excised from p2N, and the SpeI-BclI fragment wasexcised from p2Mid. Those two fragments were ligated into the NotI/BolIsites of p2CBlue in one reaction to obtain p2NMidCBlue, containing theentire PAPP-A2 cDNA. The NotI-ApaI fragment of pBluescriptIISK+ wasexcised and ligated into the NotI/ApaI sites of the mammalian expressionvector pcDNA3.1+ (Invitrogen) to obtain a modified version of thisvector, pcDNA-NA. The full length cDNA was then excised from p2NMidCBluewith NotI and XhoI and cloned into pcDNA-NA to obtain pPA2. Allrestriction sites used are in the multi cloning sites of the vectors,except for SpeI and BolI, both located in each of the two overlappingregions of the coding PAPP-A2 sequence stretches of p2N, p2Mid, and p2C(nt. 2365 and nt. 4203, respectively, of FIG. 3).

Construction of pPA2-KO: The construct pPA2-KO is a variant of the pPA2expression construct in which residue Glu-734 of the active site ofPAPP-A2 was substituted with a Gln residue. Thus, the mutant is E734Q.The pPA2-KO construct was made by site directed mutagenesis using themethod of overlap extension PCR (Ho et al., 1989, Gene 77, 51-9) withpPA2 as the template. In brief, outer primers were5′-CGCTCAGGGAAGGACAAGGG-3′ (5′ end primer, nt. 976-995 of SEQ ID NO:1)and 5′-CTAGAAGGCACAGTCGAGGC-3′ (SEQ ID NO:14) (3′ end primer, nt.1040-1021, sequence of vector pcDNA3.1+). Overlapping internal primerswere 5′-TGTCCCACTTGATGGATCATGGTGTCGGTGTGG-3′ (SEQ ID NO:15) (nt.2210-2178 of SEQ ID NO:1, nt. 2200 not C, but G resulting in E734Q) and5′-CCATCAAGTGGGACATGTTCTGGGAC-3′ (SEQ ID NO:16) (nt. 2196-2221 of SEQ IDNO:1, nt. 2200 not G, but C resulting in E734Q). The resulting mutatedfragment was digested with XbaI and XhoI and swapped into pPA2 togenerate pPA2-KO. All PCRs were carried out with Pfu DNA polymerase(Stratagene), and all constructs were verified by sequence analysis.

Construction of pPA2-mH: Two primers (5′-GAGGGCCTGTGGACCCAGGAG-3′, nt.4906-4926 of SEQ ID NO:1, and 5′-GACGTAAAGCTTCTGATTTTCTTCTGCCTTGG-3 (SEQID NO:17)′, nt. 5373-5354 of SEQ ID NO:1, preceded by a HindIII site,AAGCTT, and nt. GACGTA to facilitate cleavage of the PCR product) wereused in a PCR with pPA2 as the template to generate a nucleotidefragment encoding the C-terminal 156 residues of PAPP-A2 with the stopcodon replaced by a HindIII site for in-frame ligation to expressionvector. In brief, the PCR product was digested with EcoRI and HindIIIand cloned into the EcoRI/HindIII sites of the vectorpcDNA3.1/Myc-His(−)A to generate pPA2C-mH. The NotI-XbaI fragment(encoding the N-terminal portion of PAPP-A2), and the XbaI-EcoRIfragment (encoding the remaining central portion of PAPP-A2) wereexcised from pPA2 and ligated in one reaction into the NotI/EcoRI sitesof pPA2C-mH. The resulting construct, pPA2-mH, encoded PAPP-A2 followedby residues KLGP (SEQ ID NO:18), the myc epitope (EQKLISEEDL (SEQ IDNO:19)), residues NSAVD (SEQ ID NO:20), and six H-residues (amino acidsare given as one letter code). A stop codon follows immediately afterthe six histidine residues.

Construction of pPA2-KO-mH: A variant of pPA2-mH was constructed withresidue Glu-734 substituted into a Gln residue: The NotI-KpnI fragmentof pPA2-KO was excised and swapped into the NotI-KpnI sites of pPA2-mH,to generate pPA2-KO-mH.

Expression in mammalian cells: All constructs (pPA2, pPA2-KO, pPA2-mH,and pPA2-KO-mH) as well as empty expression vectors (pcDNA3.1+ andpcDNA3.1/Myc-His(−)A) were transiently transfected into mammalian cellsfor expression of recombinant PAPP-A2 protein. Briefly, human embryonickidney 293T cells (293tsA1609neo) (DuBridge et al., 1987, Mol Cell Biol7, 379-87) were maintained in high glucose DMEM medium supplemented with10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, andgentamicin (Life Technologies). Cells were plated onto 6 cm tissueculture dishes, and were transfected 18 h later by calcium phosphatecoprecipitation (Pear et al., 1993, Proc Natl Acad Sci USA 90, 8392-6)using 10 μg of plasmid DNA prepared by QIAprep Spin Kit (Qiagen). Aftera further 48 h the supernatants were harvested, and replaced byserum-free medium (293 SFM II, Life Technologies) for another 48 h. Theserum-free medium was harvested and cleared by centrifugation.

Analysis by Western blotting of recombinant protein resulting fromtransfection with the constructs pPA2-mH and pPA2-KO-mH, demonstratedthat PAPP-A2 is secreted as a protein of 220 kDa (See FIG. 2). Reductionof disulfide bonds did not cause a visible change in band migration.Thus, in contrast to PAPP-A, PAPP-A2 is secreted as a monomer.

Example 5 Purification by Affinity Chromatography of Tagged PAPP-A2

A metal chelate affinity column (2 ml, Pharmacia) was charged withnickel ions and loaded with serum-free medium (50 ml) from cellstransiently transfected with pPA2-KO-mH (see example 6.4). After washingin PBS containing 1M NaCl, bound protein was eluted with 10 mM EDTA inPBS in fractions of 0.5 ml. PAPP-A2 containing fractions were located bySDS-PAGE (FIG. 4, lane 5). This protein was not seen from medium ofcells transfected with empty vector (mock transfectants) and treated ina parallel manner.

Example 6 N-Terminal Sequence Analysis of PAPP-A2

C-terminally tagged PAPP-A2 purified from medium of cells transfectedwith construct pPA2-KO-mH (see examples 6.4 and 6.5) was reduced and runon a 10-20% SDS gel, and further blotted onto PVDF membrane (ProBlott,Applied Biosystems). Bands of 4 lanes were excised and subjected toN-terminal sequence analysis on an Applied Biosystems 477A sequencerequipped with an on-line HPLC (Sottrup-Jensen, 1995, Anal Biochem 225,187-8). The N-terminal sequence observed at a level of approximately 20pmol was: Ser-Pro-Pro-Glu-Glu-Ser-Asn (SPPEESN (residues 234-240 of SEQID NO:2)), resulting from cleavage before Ser-234 of the PAPP-A2polypeptide after R(230)VKK (residues 230-233 of SEQ ID NO:2).

This confirms the prediction, that PAPP-A2, like PAPP-A, is synthesizedas a prepro protein. The absence of an arginine residue in the P1position, indicates that the proprotein processing enzyme responsiblefor this cleavage is not furin, but likely another proprotein convertase(Nakayama, 1997, Biochem J 327, 625-35). Cleavage of proPAPP-A2 mighthave been predicted after R(196)QRR, which archetypically marks furincleavage (Nakayama, 1997, Biochem J 327, 625-35). We cannot exclude thatcleavage occurred at this site, and that the observed N-terminus resultsfrom further processing.

Example 7 Cleavage of Insulin-Like Growth Factor Binding Protein(IGFBP)-5

Ligand blotting (Conover et al., 1993, J Clin Invest 91, 1129-37) withradiolabeled IGF-II (Bachem) was used to assay for activity againstIGFBP-1 (from HepG2 conditioned medium), rIGFBP-2 (GroPep), rIGFBP-3(gift of D. Powell), rIGFBP-4 (Austral), rIGFBP-5 (gift of D. Andress),and rIGFBP-6 (Austral). Of the six binding proteins, IGFBP-5 showedcomplete cleavage (FIG. 5). IGFBP-3 was partially degraded (FIG. 5).This cleavage was independent of the presence of IGF. Experiments werecarried out with media from cells transfected with pPA2 or empty vector.

For further analysis, recombinant IGFBP-5 was produced in mammaliancells. In brief, human placental oligo-dT primed cDNA (Overgaard et al.,1999, Biol Reprod 61, 1083-9) was used as a template to amplify cDNAencoding human IGFBP-5 (Accession number M65062). Specific primerscontaining an XhoI site (5′-TCCGCTCGAGATGGTGTTGCTCACCGCGGT-3′ (SEQ IDNO:21)) and a HindIII site (5′-CGATAAGCTTCTCAACGTTGCTGCTGTCG-3′ (SEQ IDNO:22)) were used, and the resulting PCR product was digested and clonedinto the XhoI/HindIII sites of pcDNA3.1/Myc-His(−)A (Invitrogen). Theconstruct encoded the full-length proIGFBP-5, immediately followed byresidues KLGP, the myc epitope (EQKLISEEDL (SEQ ID NO:19)), residuesNSAVD (SEQ ID NO:20), and six H-residues (amino acids are given as oneletter code). The construct was verified by sequence analysis. PlasmidDNA for transfection was prepared by QIAprep Spin Kit (Qiagen). Cellculture and expression of recombinant IGFBP-5 was performed as describedabove in Example 6.4.

Cleavage analysis was performed by Western blotting (FIG. 6). Briefly,recombinant IGFBP-5 as contained in 5 microL cell culture medium wasincubated with culture supernatants (10 microL) from cells transfectedwith pPA2, pPA2-KO, or empty expression vectors (see example 6.4).Phosphate buffered saline was added to a final volume of 50 microL.After incubation at 37 degrees Celsius for 12 hours, 15 microL of thereaction mixture was separated by reducing 16% SDS-PAGE, blotted onto aPVDF membrane, and the C-terminal cleavage product was detected withmonoclonal anti-c-myc (clone 9E19, ATTC) using peroxidase-conjugatedsecondary antibodies (P260, DAKO), and enhanced chemiluminescence (ECL,Amersham).

Example 8 Inhibition of the Activity of PAPP-A2

Various agents were analyzed for their ability to inhibit theproteolytic activity of PAPP-A2 against IGFBP-5. The experimentalconditions were essentially as described in Example 6.7, except theagents to be tested were added (FIG. 6). Agents found to have no effecton the proteolytic activity of PAPP-A2 further included PMSF andaprotinin.

Example 9 Identification of the Cleavage Site in IGFBP-5

For cleavage site determination, purified rIGFBP-5 (FIG. 6, lane 7) wasdigested with purified PAPP-A2 and analyzed by SDS-PAGE (FIG. 6, lane8). Edman degradation of blotted material showed that both distinct,visible degradation products (FIG. 6, lane 8) contained the N-terminalsequence K(144)FVGGA (SEQ ID NO:23) (IGFBP-5 is numbered with theN-terminal Leu of the mature protein as residue 1). The two bands bothrepresent intact C-terminal cleavage fragments, because they alsocontain the C-terminal c-myc tag (FIG. 6, lane 9); they are likely to bedifferently glycosylated, in accordance with the heterogeneity ofpurified rIGFBP-5 (FIG. 6, lane 7). Both bands contained a secondsequence at lower level (45%), L(1)GXFVH (SEQ ID NO:24), correspondingto the N-terminal sequence of IGFBP-5. The absence of Ser, expected inthe third cycle, was taken as evidence for carbohydrate substitution ofSer-3. O-linked glycan on the N-terminal cleavage fragment is likely tocause it to smear around the two distinct, C-terminal fragments.Sequence analysis on the reaction mixture (>100 pmol) without SDS-PAGEseparation showed only the same two IGFBP-5 sequences in equimolaramounts. Thus, PAPP-A2 cleaves IGFBP-5 at one site, between Ser-143 andLys-144.

Example 10 Tissues where PAPP-A2 may Cause Proteolysis of IGFBP-5

Proteolyctic activity against IGFBP-5 has been widely reported fromseveral sources, e.g. pregnancy serum (Claussen et al., 1994,Endocrinology 134, 1964-6), seminal plasma (Lee et al., 1994, J ClinEndocrinol Metab 79, 1367-72), culture media from smooth muscle cells(Imai et al., 1997, J Clin Invest 100, 2596-605), granulosa cells(Resnick et al., 1998, Endocrinology 139, 1249-57), osteosarcoma cells(Conover and Kiefer, 1993, J Clin Endocrinol Metab 76, 1153-9), and alsofrom osteoblasts (Thrailkill et al., 1995, Endocrinology 136, 3527-33),and fibroblasts (Busby et al., 2000, J Biol Chem). In general, theproteinase responsible for cleavage of IGFBP-5 has remainedunidentified.

The recent identification of PAPP-A as the IGFBP-4 proteinase infibroblasts and osteoblasts (Lawrence et al., 1999, Proc Natl Acad SciUSA 96, 3149-53), ovarian follicular fluid (Conover et al., 1999, J ClinEndocrinol Metab 84, 4742-5), pregnancy serum (Overgaard et al., 2000, JBiol Chem), and vascular smooth muscle cells (Bayes-Genis, A., Schwartz,R. S., Ashai, K., Lewis, D. A., Overgaard, M. T., Christiansen, M.,Oxvig, C., Holmes, D. R., Jr., and Conover, C. A. Arterioscler. Thromb.Vasc. Biol., in press) firmly establishes PAPP-A and IGFBP-4 as animportant functional pair in several systems. No other substrate as hasbeen found for PAPP-A, and no other proteinase has been shown to cleaveIGFBP-4 physiologically. It is therefore likely that the pair of PAPP-A2and IGFBP-5 plays an analogous role in a number of the tissues mentionedabove and/or elsewhere. Interestingly, incubating IGFBP-5 with smoothmuscle cells conditioned medium resulted in cleavage between Ser-143 andLys-144 (Imai et al., 1997, J Clin Invest 100, 2596-605), the samecleavage site as found here with PAPP-A2. This immediately suggestsPAPP-A2 as an obvious candidate IGFBP-5 proteinase for this tissue.

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That which is claimed is:
 1. A cDNA that encodes a polypeptide that (a)consists of amino acid residues 234-1791 of SEQ ID NO: 2 (maturepregnancy associated plasma protein A2 (PAPP-A2)); or (b) is at least95% sequence identical to the polypeptide of (a), and differs from thepolypeptide of (a) solely by (i) deletion of 1-10 amino acid residuesfrom, or addition of 1-10 residues to, the amino terminal, and/or (ii)deletion of 1-10 amino acid residues from, or addition of 1-10 residuesto, the carboxy terminal, and/or (iii) one or more conservativesubstitutions; wherein said polypeptide has a proteolytic activityagainst Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).
 2. ThecDNA of claim 1, wherein the cDNA encodes the polypeptide according to(a).
 3. The cDNA of claim 1, wherein the cDNA encodes the polypeptideaccording to (b).
 4. The cDNA of claim 3, wherein the amino acidsequence of said polypeptide according to (b) differs from that of thepolypeptide according to (a) solely by one or more conservativesubstitutions.
 5. An isolated polynucleotide comprising nucleotides1-5376 of SEQ ID NO:
 1. 6. An isolated polynucleotide comprising thecDNA of claim 1 operably linked to a polynucleotide encoding (a) animmunogenic carrier protein, and/or (b) a tag that facilitates thedetection or purification of the polypeptide encoded by said isolatedpolynucleotide.
 7. The cDNA of claim 1, 2, 3, 4 or 6 or thepolynucleotide of claim 5, wherein the cDNA or the polynucleotide isoperably linked to a further polynucleotide comprising nucleotides 5377to 8527 of SEQ ID NO: 1, corresponding to a 3′ untranslated region. 8.An isolated polynucleotide encoding a polypeptide that (a) consists ofamino acid residues 234-1791 of SEQ ID NO: 2 (mature pregnancyassociated plasma protein A2 (PAPP-A2)); or (b) is at least 95% sequenceidentical to the polypeptide of (a), and differs from the polypeptide of(a) solely by (i) deletion of 1-10 amino acid residues from, or additionof 1-10 residues to, the amino terminal, and/or (ii) deletion of 1-10amino acid residues from, or addition of 1-10 residues to, the carboxyterminal, and/or (iii) one or more conservative substitutions; whereinsaid polypeptide has a proteolytic activity against Insulin Like GrowthFactor Binding Protein 5 (IGFBP-5), and wherein said polynucleotide isoperably linked to a polynucleotide having the sequence of nucleotides5377-8527 of SEQ ID NO:
 1. 9. An expression vector comprising thesequence of a polynucleotide encoding a polypeptide that (a) consists ofamino acid residues 234-1791 of SEQ ID NO:2 (mature pregnancy associatedplasma protein A2 (PAPP-A2)); or (b) is at least 95% sequence identicalto the polypeptide of (a), and differs from the polypeptide of (a)solely by (i) deletion of 1-10 residues from, or addition of 1-10residues to, the amino terminal, and/or (ii) deletion of 1-10 residuesfrom, or addition of 1-10 residues to, the carboxy terminal, and/or(iii) one or more conservative substitutions; wherein said polypeptidehas a proteolytic activity against Insulin Like Growth Factor BindingProtein 5 (IGFBP-5).
 10. An isolated host cell comprising the vector ofclaim
 9. 11. A method for expression of a polynucleotide in arecombinant host cell, the polynucleotide encoding a polypeptide that(A) consists of amino acid residues 234-1791 of SEQ ID NO: 2 (maturepregnancy associated plasma protein A2 (PAPP-A2)); or (B) is at least95% sequence identical to the polypeptide of (A), and differs from thepolypeptide of (A) solely by (i) deletion of 1-10 amino acid residuesfrom, or addition of 1-10 residues to, the amino terminal, and/or (ii)deletion of 1-10 amino acid residues from, or addition of 1-10 residuesto, the carboxy terminal, and/or (iii) one or more conservativesubstitutions; wherein said polypeptide has a proteolytic activityagainst Insulin Like Growth Factor Binding Protein 5 (IGFBP-5), themethod comprising the steps of (a) providing said polynucleotide, anexpression vector, and a recombinant host cell, (b) cloning thepolynucleotide into the expression vector, (c) transforming therecombinant host cell with the expression vector obtained in (b), and(d) expressing the polypeptide in the recombinant host cell obtained in(c).